Methods for treating neurological conditions and exposure to nerve agents

ABSTRACT

Described are methods of treating or reducing the toxic effects of exposure to a nerve agent, comprising administering to a subject in need thereof (i) an AMPA/GluR5(GluK1) kainate receptor antagonist (such as LY293558) and (ii) an NMD A receptor antagonist (such as an antimuscarinic compound, such as caramiphen), as well as methods of treating, reducing the risks of, or preventing a neurological condition such as epilepsy, seizures, post-traumatic stress disorder, status epilepticus, depression, or anxiety, comprising administering to a subject in need thereof (i) an AMPA/GluR5(GluK1) kainate receptor antagonist (such as LY293558) and (ii) an NMDA receptor antagonist (such as an antimuscarinic compound, such as caramiphen). The methods may further comprise administering a positive allosteric modulator of synaptic GABA A  receptors, such as a benzodiazepine, such as midazolam, to the subject. The methods are suitable for use in children and adults. Related compositions and uses also are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefits of U.S. ProvisionalApplication No. 62/349,819, filed Jun. 14, 2016, the contents of whichare hereby incorporated by reference in their entirety.

GOVERNMENT RIGHTS

This invention was made with government support under NS094131 awardedby National Institutes of Health. The government has certain rights inthe invention.”

FIELD

The present disclosure relates generally to the field of treatingneurological conditions, including neurological conditions triggered byexposure to nerve agents, including seizures. Described are methodscomprising administering to a subject in need thereof (i) at least oneAMPA/GluR5 (GluK1) kainate receptor antagonist and (ii) at least oneNMDA receptor antagonist. In some embodiments, the methods furthercomprise administering a positive allosteric modulator of synapticGABA_(A) receptor), such as a benzodiazepine, such as midazolam. Theneurological conditions that can be treated include, but are not limitedto epilepsy, seizures (e.g., refractory seizures; seizures caused byexposure to nerve agents, such as organophosphorus nerve agentsincluding soman and sarin; seizures caused by chemical (e.g. alcohol oropiate) withdrawal; etc.), post-traumatic stress disorder (PTSD), statusepilepticus (SE), depression, and anxiety.

BACKGROUND

The following discussion is merely provided to aid the reader inunderstanding the disclosure and is not admitted to describe orconstitute prior art thereto.

Many neurological conditions, such as epilepsy, seizures, post-traumaticstress disorder (PTSD), status epilepticus (SE), depression, and anxietyare caused by aberrant chemical signaling in the brain or centralnervous system (CNS). In particular, signaling throughα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAreceptors), kainate receptors (KARs, which are ionotropic receptors madeup of five types of subunits including GluR5(GluK1), GulR6, GluR7, KA1,and KA2), and N-methyl-D-aspartate receptor (NMDA receptors) can play arole in each of these conditions. Nerve agents and otherorganophosphorus compounds may interfere with these same signalingpathways.

Nerve agents such as organophosphorus nerve agents are potent toxinsthat act primarily by inhibiting the activity of acetylcholinesterase.The resulting accumulation of acetylcholine at synaptic junctionsproduces peripheral cholinergic crisis (excessive salivation,lacrimation, rhinorrhea, bronchorrhea, cardiorespiratory suppression,eventual muscle paralysis, etc.), and induces seizures and statusepilepticus (SE), resulting in brain damage and death. The possibilitythat nerve agents can be employed by terrorists to inflict masscasualties necessitates readiness on the part of the government toprotect the people, including the more vulnerable sections of thepopulation, including children. The devastating effects of the sarinattack in Syria in August of 2013, where 1,400 civilians were killed,426 of which were children, underscored the need for effectivetreatments to save lives and protect against long-term healthconsequences of exposure to nerve agents.

It is generally understood that during seizures, including seizuresinduced by nerve agents, GABAergic and glutamatergic activity are out ofbalance, with a strong dominance of the latter. Without wanting to bebound by theory, it is believed that initiation of seizures (e.g.,seizures caused by nerve agent exposure) is due to excessive elevationof acetylcholine—secondarily to AChE inhibition—acting primarily onmuscarinic receptors, but that seizures are sustained and reinforced byglutamatergic rather than cholinergic mechanisms.

One way to suppress glutamatergic hyperactivity is by enhancingGABAergic inhibition. Accordingly, benzodiazepines, which are positiveallosteric modulators of GABA_(A) receptors, have long been the firstline of treatment to induce cessation of SE triggered by variousetiologies, including nerve agents. The benzodiazepine diazepam (DZP) iscurrently the only FDA-approved injectable drug for the control ofseizures caused by nerve agents. However, the efficacy ofbenzodiazepines decreases significantly as the time between initiationof SE and treatment increases. This represents a significant drawback inthe context of SE and other conditions involving unpredictable seizures,including seizures induced by nerve agents, because it may be difficultor impossible to administer treatment promptly, such as after exposureto nerve agents caused by a terrorist attack or after anotherseizure-triggering event.

Benzodiazepines also may become less effective over time, e.g., atolerance to benzodiazepines may develop, in which case seizures mayrecur. Additionally, while DZP has been used for adults, it may not besuitable for use in children. For example, there is evidence suggestingthat immature animals differ from adult animals both in seizuresusceptibility and in the extent and nature of neuropathology thatseizures can induce. The American Academy of Pediatrics have announcedthat mass casualties during terrorist attacks that employ nerve agentsare expected to disproportionately affect children due to their greaterbody surface area-to-body mass ratio, increased skin permeability,faster respiration rate, breathing at a level where nerve agent vapordensity would be highest, and an increased susceptibility to seizures.Although the American Academy of Pediatrics has identified reasons whychildren are more vulnerable to nerve agent toxicity, there is verylittle information on the appropriate countermeasures for use in thepediatric population.

Aberrant biochemical signaling in the brain and CNS also can lead toepilepsy, depression, anxiety, and related conditions. Hyperexcitabilityof the amygdala is a characteristic of depression, anxiety disorders,and PTSD, and hyperexcitability of the basolateral nucleus of theamygdala (BLA) may be particularly important in this regard. The BLA hasa remarkably high expression of GluK1 kainate receptors (GluK1KRs),which can impact the modulation of GABAergic and glutamatergictransmission in the BLA. Indeed, the net effect of GluK1KR activation isto increase excitability, and therefore blockade of GluK1KRs in the BLAcan produce anxiolytic effects. Likewise, depressive patients are knownto respond to NMDAR antagonists and there is some evidence in ratssuggesting that inhibition of this receptor can produce antidepressanteffects. Yet, depression, anxiety, and anxiety-related disorderscontinue to be a pressing problems in the health system of the UnitedStates and abroad.

Accordingly, there is a need for effective methods of treatingneurological conditions, such as epilepsy, various types of seizures,post-traumatic stress disorder (PTSD), status epilepticus, depression,and anxiety, including methods that may be suitable for use in children.

SUMMARY

Described herein are methods for treating neurological conditions,including methods for treating or reducing the toxic effects of exposureto nerve agents.

For instance, in one aspect, the present disclosure relates to methodsof treating or reducing the toxic effects of exposure to a nerve agent,comprising administering to a subject in need thereof (i) anAMPA/GluR5(GluK1) kainate receptor antagonist and (ii) an NMDA receptorantagonist.

In another aspect, the present disclosure relates to methods of treatingor reducing the risks of a neurological condition, comprisingadministering to a subject in need thereof (i) an AMPA/GluR5(GluK1)kainate receptor antagonist and (ii) an NMDA receptor antagonist. Insome embodiments, the neurological condition may be, for example,epilepsy, seizure, post-traumatic stress disorder, status epilepticus,depression, or anxiety.

In some embodiments, the AMPA/GluR5(GluK1) kainate receptor antagonistis LY293558.

In some embodiments, the NMDA receptor antagonist is an antimuscariniccompound like, for example, caramiphen.

In some embodiments, the disclosed methods may further compriseadministering a positive allosteric modulator of synaptic GABA_(A)receptors to the subject. In some embodiments, the positive allostericmodulator of synaptic GABA_(A) receptors is a benzodiazepine, forexample, midazolam.

In some embodiments, the AMPA/GluR5(GluK1) kainate receptor antagonistand the NMDA receptor antagonist, and, optionally, the positiveallosteric modulator of synaptic GABA_(A) receptors, are administered inthe same composition.

In some embodiments, the AMPA/GluR5(GluK1) kainate receptor antagonistand the NMDA receptor antagonist, and, optionally, the positiveallosteric modulator of synaptic GABA_(A) receptors, are administered inseparate compositions, by the same route of administration or bydifferent routes of administration. For instance, the AMPA/GluR5(GluK1)kainate receptor antagonist and the NMDA receptor antagonist, and,optionally, the positive allosteric modulator of synaptic GABA_(A)receptors, are administered substantially simultaneously. Alternatively,the AMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, are administered sequentially.

In some embodiments, the AMPA/GluR5(GluK1) kainate receptor antagonistis administered prior to the administration of the NMDA receptorantagonist. In some embodiments, the AMPA/GluR5(GluK1) kainate receptorantagonist is administered after to the administration of the NMDAreceptor antagonist. In some embodiments, the positive allostericmodulator of synaptic GABA_(A) receptors is administered prior to boththe AMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, after both the AMPA/GluR5(GluK1) kainate receptor antagonistand the NMDA receptor antagonist, or prior to one of theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist and after the other of the AMPA/GluR5(GluK1) kainate receptorantagonist and the NMDA receptor antagonist.

In some embodiments, the AMPA/GluR5(GluK1) kainate receptor antagonistand the NMDA receptor antagonist and, optionally, the positiveallosteric modulator of synaptic GABA_(A) receptors, are administered byinjection like, for example, an intramuscular injection. In someembodiments, the AMPA/GluR5(GluK1) kainate receptor antagonist and theNMDA receptor antagonist and, optionally, the positive modulator ofsynaptic GABA-A receptors, are administered orally.

In some embodiments relating to treating or reducing the toxic effectsof exposure to a nerve agent, the nerve agent comprises anorganophosphorus toxin, such as soman or sarin. In some embodiments, thesubject has been exposed to a nerve agent. In some embodiments,administration of the AMPA/GluR5(GluK1) kainate receptor antagonist andNMDA receptor antagonist occurs within 20 minutes or less, one hour orless, or two hours or less of exposure to the nerve agent. In someembodiments, the subject is suspected of having been exposed to a nerveagent. In some embodiments, the subject is at risk of exposure to anerve agent. In some embodiments, the method is effective to treat orreduce the toxic effects of exposure to the nerve agent selected fromone or more of seizures, status epilepticus, brain damage, neurologicaleffects, behavioral effects, difficulty breathing, nausea, loss ofcontrol of bodily functions, and death.

In some embodiments related to treating or reducing the risks of aneurological condition, the neurological condition is a seizure such asa refractory seizure, an epileptic seizure, or a seizure induced bywithdrawal from a chemical (e.g. alcohol or opiate). For example, whenthe seizure is induced by withdrawal from a chemical (e.g. alcohol oropiate), administration of the AMPA/GluR5(GluK1) kainate receptorantagonist and NMDA receptor antagonist, and, optionally, the positiveallosteric modulator of synaptic GABA_(A) receptors, may occur prior towithdrawal of the chemical, during detoxification of the subject, orafter withdrawal of the chemical. In some embodiments, the subject hasbeen diagnosed with the neurological condition. In some embodiments, themethod is effective to treat or reduce signs or symptoms of theneurological condition.

In some embodiments, the disclosed methods further comprise at least onesubsequent administration of the AMPA/GluR5(GluK1) kainate receptorantagonist and NMDA receptor antagonist and, optionally, the positiveallosteric modulator of synaptic GABA_(A) receptors. In someembodiments, the at least one subsequent administration is administeredby injection, while in some embodiments, the at least one subsequentadministration is administered orally.

In embodiments of any of the methods described herein, the subject maybe a mammal, such as a human child or a human adult.

Also provided are an AMPA/GluR5(GluK1) kainate receptor antagonist andan NMDA receptor antagonist, and, optionally a positive allostericmodulator of synaptic GABA_(A), as described herein, for treating orreducing the toxic effects of exposure to a nerve agent.

Also provided are an AMPA/GluR5(GluK1) kainate receptor antagonist andan NMDA receptor antagonist, and, optionally a positive allostericmodulator of synaptic GABA_(A), as described herein, for treating orreducing the risks of a neurological condition.

Also provided are uses of (i) an AMPA/GluR5(GluK1) kainate receptorantagonist and (ii) an NMDA receptor antagonist, and, optionally apositive allosteric modulator of synaptic GABA_(A), as described herein,in the preparation of one or more medicaments as described herein fortreating or reducing the toxic effects of exposure to a nerve agent asdescribed herein, or for treating or reducing the risks of aneurological condition as described herein.

The foregoing general description and following detailed description areexemplary and explanatory and not limiting of the invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-D show the effect of the methods described herein in a ratmodel, using LY293558 as the AMPA/GluR5(GluK1) kainate receptorantagonist and caramiphen (CRM) as the NMDA receptor antagonist to stopseizures induced by the nerve agent soman (1.2×LD₅₀, 132 μg/kg), wherethe method reduced the duration of the initial SE and the total durationof SE as compared to treatment with LY293558 alone. FIG. 1A shows thetime course of seizure suppression by LY293558, while FIG. 1B shows thetime course of seizure suppression by LY293558+CRM. FIG. 1C illustrateselectrode placement (1, 2, 3, 4) where electrical activity was sampled(1: left frontal; 2: right frontal; 3: left parietal; 4: right parietal;un-labeled: cerebellar reference electrode). FIG. 1D, left panel, showsthe duration of the initial SE, which is the SE that started within 6-10min after soman exposure and was terminated by 15 mg/kg LY293558 (openbar), or 15 mg/kg LY293558 and 50 mg/kg CRM (closed bar). FIG. 1D, rightpanel, shows the duration of SE throughout the 7 day-period after somanexposure for the same two groups. The corresponding durations for theuntreated soman group (not shown) are about 700 min for the initial SEand more than 1000 min for the 7-day-period. *p<0.05 (Unequal Variancet-test).

FIGS. 2A-D show that the methods described herein are superior totreatment with LY293558 alone in preventing soman-induced neuronaldegeneration 7 days after exposure in a rat model. FIGS. 2A and 2B showpanoramic photomicrographs of Nissl-stained sections showing the brainregions from the Fluoro-Jade C photomicrographs displayed in FIG. 2C.FIG. 2C shows representative photomicrographs of Fluoro-Jade C stainedsections from the brain regions where neuronal degeneration wasevaluated, for the untreated (SOMAN), LY293558-treated (SOMAN+LY293558)and LY293558- and CRM-treated (SOMAN+LY293558+CRM) groups. Totalmagnification is 100×. Scale bar is 50 μm. FIG. 2D shows medianneuropathology score and interquartile range for the amygdala (Amy),piriform cortex (Pir), entorhinal cortex (Ent), the CA1 and CA3subfields of the ventral hippocampus, hilus, and neocortex (neo-Ctx).

FIG. 3 shows results of weight loss study during 7 days after somanexposure in a rat model. Soman-exposed rats that did not receiveanticonvulsant treatment (n=18) display dramatic weight loss and reducedweight gain. Rats treated with LY293558 only (n=18) exhibitedsignificantly improved weight-gain response, while rats treated withLY293558+CRM (n=19) do not display any difference from control rats(n=18) that were not exposed to soman.

FIG. 4A-4C includes a schematic diagram of a study protocol and resultsin a rat model, showing that treatment with LY293558 alone or bothLY293558 and CRM, but not CRM alone, prevented increase in anxiety 1month after soman-induced SE. Similar results were obtained at the 3month time point (not shown). FIG. 4A shows time spent in the center ofthe open field, which was significantly reduced only in the grouptreated with CRM alone. FIG. 4B shows that there was no effect on thetotal distance travelled. FIG. 4C shows that acoustic startle responsesto both 110 dB and 120 dB acoustic stimuli were significantly increasedonly in the group treated with CRM alone. *p<0.05 compared to Control(One Way ANOVA, Dunnett post-hoc), n=8˜12 rats/group.

FIGS. 5A-5B show increased survival rate and seizure control in animalstreated by the present methods. FIG. 5A shows that treatment withLY293558 and CRM significantly increased the survival rate of 21 day oldrats exposed to 1.2×LD₅₀ of soman (74.4 μg/kg), as compared to animalstreated with only one of LY293558 or CRM. FIG. 5B shows that treatmentwith LY293558 and CRM significantly reduced the time to stop seizures in21 day old rats exposed to 1.2×LD₅₀ of soman, as compared to animalstreated with only one of LY293558 or CRM.

FIG. 6 shows that treatment with LY293558 and CRM reduces the severityof soman induced seizures faster than either compound alone in a ratmodel. CRM alone, at a dose of 50 mg/kg, was not effective in reducingseizure severity within 120 min after exposure.

FIG. 7A-7B show the effects of soman exposure on the amygdala 30 daysand 3 months after exposure in a rat model. FIG. 7A shows tracings ofthe amygdala in series of slices (left) and representativephotomicrographs (middle) from control animals (n=7), soman-exposedanimals that received CRM (50 mg/kg) at 60 minutes post-exposure (n=10),soman-exposed animals that received LY293558 (20 mg/kg) at 60 minutesafter soman-injection (n=10) and soman-exposed animals that receivedboth LY293558 and CRM at 60 minutes after soman-injection (n=10). FIG.7B shows group data showing the estimated volume of the amygdala for allthree groups, 30 days after the exposure (top) and 3 months after theexposure (bottom). * p<0.05.

FIGS. 8A-8B show the effects of soman exposure on the hippocampus 30days and 3 months after exposure in a rat model. FIG. 8A shows tracingsof the hippocampus in series of slices (left) and representativephotomicrographs (right) from control animals (n=7), soman-exposedanimals that received CRM (n=7), and soman-exposed animals that receivedLY293558 (20 mg/kg) at 1 hour after soman injection (n=8). FIG. 8B showsgroup data showing the estimated volume of the hippocampus for all threegroups, 30 days after the exposure, and group data showing the estimatedvolume of the hippocampus for all three groups, 90 days after theexposure. *p<0.05.

FIG. 9 shows that treatment with LY293558 and CRM reduced anxiety inopen field and acoustic startle response tests 30 days after exposure tosoman in a rat model, as shown by % time spent in the center of the openfield, distance travelled, and Startle Response Amplitude, for thecontrol group (n=14), soman-exposed rats who received CRM (n=16),similarly treated rats who received LY293558 at 60 minutes after somanexposure (n=13), and rats who received both LY293558 and CRM (n=14).

FIG. 10 shows that treatment with LY293558 and CRM reduced anxiety inopen field and acoustic startle response tests 3 months after exposureto soman, as shown by: % time spent in the center of the open field anddistance travelled, and Startle Response Amplitude, for the controlgroup (n=12), soman-exposed rats who received CRM (n=13), similarlytreated rats who received LY293558 at 60 minutes after soman exposure(n=13), and for rats who received combination of LY293558 and CRM(n=12).

FIGS. 11A-11B show that treatment with LY293558 and CRM prevented areduction in charge transferred by GABA_(A) receptor-mediatedspontaneous IPSCs s recorded from principal cells of the basolateralamygdala, 30 days (FIG. 11A) and 3 months (FIG. 11B) after exposure tosoman. The left panels show example current traces in v-clamp mode.GABAaRs-mediated sIPSCs were recorded at +30 mV holding potential. Theright panels show charge [pico-Coulombs; pC] transferred by sIPSCs ofBLA principal neurons during a 40 second time window. For the 30 daytime point: Control: 413.23±84.19 (n=7); CRM treated: 201.49±30.91(n=7); LY293558 treated: 359.1±53.98 (n=8); LY293558+CRM treated:402.57±85.86 (n=13). * p<0.05 compared to Control (independent t-test).For the 3 month time point: Control: 669.94±124.59 (n=10); CRM treated:378.68+-40.67 (n=7) ; LY293558-treated: 627.89±119.90 (n=7); bothLY293558- and CRM-treated: 683.22±136.41 (n=6). * p<0.05 compared toControl (independent t test).

FIG. 12 shows suppression of initial status epilepticus (SE) withLY293558 and significant suppression over 24 hours. Soman (n=4) andsoman+LY293558 (n=7). ** p<0.01 and *** p<0.001 by unequal variancet-test.

FIG. 13 shows protection against neuronal damage in the subcortex and aneocortical sample region by LY293558 administration, in rats studied atday 7 following soman exposure. Soman (n=6) and soman+LY293558 (n=6).Subcortical regions evaluated include amygdala (Amy), piriform cortex(PIR), neocortex (Neo-Ctx), CA1 and CA3 regions of the hippocampus, thehilus and entorhinal cortex (Ent). * p<0.05 and ** p<0.01 byMann-Whitney U test.

FIG. 14 shows suppression of initial status epilepticus (SE) withLY293558 and significant suppression over 24 hours. Soman (n=5) andsoman+LY293558 (n=8). ** p<0.01 and *** p<0.001 by unequal variancet-test.

FIG. 15 shows plasma and brain levels of LY293558 following 15 mg/kgIntramuscular injection. Plasma levels (squares) and brain levels(triangles) of LY293558 following 15 mg/kg, intramuscular. Plasmasamples sizes are n=3-6 per time point and brain samples are n=3 pertime point. Data are mean +/−standard deviation.

FIG. 16 shows protection against neuronal damage in the subcortex and aneocortical sample region, with LY293558 in rats studied at day 7following soman exposure. Soman (n=18) and soman+LY293558 (n=18). Brainregions evaluated include amygdala (Amy), piriform cortex (PIR),neocortex (Neo-Ctx), CA1 and CA3 regions of the hippocampus, the hilusand entorhinal cortex (Ent). * p<0.05, ** p<0.01 and ** p<0.001 byMann-Whitney U test.

FIGS. 17A-17B show suppression of initial status epilepticus (SE) withdiazepam (DZP) or UBP302 administered at 1 or 2 hours post somanexposure and significant suppression for UBP302 over 24 Hours. FIG. 17Ashows observations of total SE over 24 hours, for soman (n=4), soman+DZP(n=6) and soman+UBP302 (n=8). FIG. 17B shows observations of total SEover 24 hours, for soman (n=4), soman+DZP (n=4) and soman+UBP302 (n=4),*p<0.05, ** p<0.01 and *** p<0.001 by ANOVA and post-hoc testing.

FIG. 18 shows protection against neuronal damage in the subcortex and aneocortical brain region with UBP302, and no observed neuroprotectionwith diazepam. Observations are n=6 for each group. Brain regionsevaluated include amygdala (Amy), piriform cortex (PIR), entorhinalcortex (Ent), CA1 and CA3 regions of the hippocampus, the hilus andneocortex (Neo-Ctx), * p<0.05 and ** p<0.01 by Mann-Whitney U test.

FIG. 19 shows that LY293558 produces a significantly greater reductionin the total duration of status epilepticus (SE) in the 24-h periodafter soman exposure in comparison to midazolam (MDZ). Soman+midazolam(n=4) and soman+LY293558 (n=4). * p<0.05 Student t-test.

FIGS. 20A-20C show LY293558, but not midazolam, protects againstsoman-induced long-term increases in anxiety-like behavior as well asreduced inhibitory synaptic transmission in the Basolateral Nucleus ofAmygdala (BLA), 3 months after exposure to soman. FIG. 20A and FIG. 20B:Observations are n=10 for each group. FIG. 20C: Observations are n=16for Control, n=7 for Midazolam and n=5 for LY293558. FIG. 20A: Percentof Time Spent in the Center of the Open Field test. FIG. 20B: StartleResponse amplitude measured during Acoustic Startle Response test. FIG.20C: Charge (in pico-Coulombs; pC) transferred by GABA-Areceptor-mediated spontaneous Inhibitory Postsynaptic Currents (sIPSCs)recorded from BLA principal neurons during 10 s time window. *p<0.05,One-Way ANOVA with Dunnett's T post hoc.

FIGS. 21A-21C show increased survival rate and seizure control innewborn animals treated with LY293558 and CRM. FIG. 21 A shows theexperimental protocol for newborn animals. FIG. 21B shows that treatmentwith LY293558 and CRM significantly increased the survival rate of 12day old rats exposed to 1.2×LD₅₀ of soman (32.4 μg/kg), as compared toanimals treated with only CRM. FIG. 21C shows that treatment withLY293558 and CRM significantly reduced the time to stop seizures in 12day old rats exposed to 1.2×LD₅₀ of soman, as compared to animalstreated with only one of LY293558 or CRM.

FIGS. 22A-22C show that the combination of CRM and LY293558 protectsagainst the development of epilepsy after soman exposure (1.2×LD₅₀).Representative EEG tracings (60 s duration) 7 days after exposure to1.2×LD₅₀ of soman. FIG. 22A shows an animal from SOMAN group showing aspontaneous recurrent seizure (behavioral correlates, rearing andfalling). FIG. 22B shows an animal from SOMAN+LY293558 group withpresence of spontaneous epileptiform discharges. FIG. 22C shows ananimal from SOMAN+LY293558+CRM with normal EEG.

FIGS. 23A-23C show increased anxiety in the open field and acousticstartle response tests, 30 days after soman exposure, was prevented byadministration of LY2935578 or combination of LY293558 and caramiphen inP12 rats. FIGS. 23A-23B show percent time spent in the center of theopen field (A) and distance travelled (B). FIG. 23C shows startleresponse amplitude for the control group (n=9), soman-exposed rats whoreceived CRM (n=8), similarly treated rats who received LY293558 at 60min after soman exposure (n=9), and for rats who received thecombination of LY293558 and CRM (n=9). * p<0.05 compared to Controlgroup, ANOVA Dunnett post-hoc test.

FIGS. 24A-24B show LY293558 or combination of LY293558 and caramiphenprevented the reduction in charge transferred by GABA_(A)receptor-mediated spontaneous IPSCs, 30 days after exposure to soman inP12 rats. FIG. 24A shows example current traces in v-clamp mode.GABA_(A) receptor-mediated sIPSCs were recorded at +30 mV holdingpotential. FIG. 24B shows charge [pico-Coulombs; pC] transferred bysIPSCs of BLA principal neurons during 10 s time window, 30 days aftersoman exposure. N=8˜20 cells/group ** p<0.05 compared to Control group,ANOVA LSD post-hoc Test.

FIGS. 25A-25C show increased anxiety in the open field and acousticstartle response 3 months after soman exposure were prevented byadministration of LY2935578 or combination of LY293558 and caramiphen,in P12 rats. FIGS. 25A and 25B show percent time spent in the center ofthe open field (A) and distance travelled (B). FIG. 25C shows startleresponse amplitude for the control group (n=8), soman-exposed rats whoreceived CRM (n=8), similarly treated rats who received LY293558 at 60min after soman exposure (n=9), and for rats who received thecombination of LY293558 and CRM (n=9). ** p<0.01 compared to Controlgroup, ANOVA Dunnett post-hoc test.

FIGS. 26A-26B show LY293558 or combination of LY293558 and caramiphenprevented the reduction in charge transferred by GABA_(A)receptor-mediated spontaneous IPSCs, 3 months after exposure to soman inP12 rats. FIG. 26A shows example current traces in v-clamp mode.GABA_(A) receptor-mediated sIPSCs were recorded at +30 mV holdingpotential. FIG. 26B shows charge [pico-Coulombs; pC] transferred bysIPSCs of BLA principal neurons during 10 s time window, 30 days aftersoman exposure. N=8˜20 cells/group ** p<0.05 compared to Control ANOVALSD post-hoc Test.

FIG. 27 shows a generalized study scheme for proposed efficacy studiesof treating nerve agent exposure in 21 day old rats with a combinationof LY293558, caramiphen, and midazolam.

DETAILED DESCRIPTION

Disclosed herein are methods of treating neurological conditions, andmethods for treating or reducing the toxic effects of exposure to nerveagents, including seizures. The neurological conditions that may betreated in accordance with the methods described herein includeepilepsy, various types of seizures (e.g., seizures caused by exposureto nerve agents, refractory seizures, seizures due to chemical (e.g.alcohol or opiate) withdrawal, etc.), post-traumatic stress disorder(PTSD), status epilepticus (SE), depression, and anxiety. The methodsmay comprise administering to a subject in need thereof (i) at least oneAMPA/GluR5(GluK1) kainate receptor antagonist and (ii) at least one NMDAreceptor antagonist. In some embodiments, the methods further compriseadministering a positive allosteric modulator of synaptic GABA_(A)receptors, such as a benzodiazepine, such as midazolam, to the subject.For instance, in one aspect, the present disclosure relates to methodsof treating or reducing the toxic effects of exposure to a nerve agent,comprising administering to a subject in need thereof (i) anAMPA/GluR5(GluK1) kainate receptor antagonist and (ii) an NMDA receptorantagonist. In another aspect, the present disclosure relates to methodsof treating or reducing the risks of a neurological condition, such asone or more selected from epilepsy, various types of seizures (e.g.,seizures caused by exposure to nerve agents, refractory seizures,seizures due to chemical (e.g. alcohol or opiate) withdrawal, etc.),post-traumatic stress disorder (PTSD), status epilepticus (SE),depression, and anxiety, comprising administering to a subject in needthereof (i) an AMPA/GluR5(GluK1) kainate receptor antagonist and (ii) anNMDA receptor antagonist. The AMPA/GluR5(GluK1) kainate receptorantagonist may be LY293558. Independently, the NMDA receptor antagonistmay be caramiphen (CRM). In any embodiments, the subject may further beadministered a positive allosteric modulator of synaptic GABA_(A)receptors, such as a benzodiazepine, such as midazolam. Also describedare (i) a AMPA/GluR5(GluK1) kainate receptor antagonist and (ii) an NMDAreceptor antagonist, and, optionally, a positive allosteric modulator ofsynaptic GABA_(A) receptors, for treating or reducing the toxic effectsof exposure to a nerve agent, or for treating or reducing the risks of aneurological condition. Also described are uses of such agents in thepreparation of one or more medicaments for treating or reducing thetoxic effects of exposure to a nerve agent, or for treating or reducingthe risks of a neurological condition.

In subjects exposed to or at risk of exposure to a nerve agent, themethods or uses may be effective to treat, prevent, reduce, ameliorate,or eliminate the effects of exposure to a nerve agent, such as seizures,brain damage, behavioral deficits, pathophysiological alterations inbrain regions that underlie these deficits, and death. In subjectssuffering from a neurological condition, the methods or uses may beeffective to treat, prevent, reduce, ameliorate, or eliminate theeffects of the neurological condition, such as seizures, brain damage,behavioral deficits, pathophysiological alterations in brain regionsthat underlie these deficits, and death.

The inventor discovered that treating subjects suffering from aneurological condition and/or exposed to nerve agents with both (i) anAMPA/GluR5(GluK1) kainate receptor antagonist such as LY293558 and (ii)an NMDA receptor antagonist such as CRM achieved synergistic resultswith regard to therapeutic efficacy. In the context of treating exposureto nerve agents, the described methods have been shown to be superiorlyefficacious not only in stopping seizures very rapidly, but also inpreventing brain damage, behavioral deficits, and resulting in veryquick recovery.

Definitions

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art, unless otherwisedefined. Any suitable materials and/or methodologies known to those ofordinary skill in the art can be utilized in carrying out the methodsdescribed herein.

As used in the description of the invention and the appended claims, thesingular forms “a”, “an” and “the” are used interchangeably and intendedto include the plural forms as well and fall within each meaning, unlessthe context clearly indicates otherwise. Also, as used herein, “and/or”refers to and encompasses any and all possible combinations of one ormore of the listed items, as well as the lack of combinations wheninterpreted in the alternative (“or”).

As used herein, the term “about” will be understood by persons ofordinary skill in the art and will vary to some extent depending uponthe context in which it is used. If there are uses of the term which arenot clear to persons of ordinary skill in the art given the context inwhich it is used, “about” will mean up to plus or minus 10% of theparticular term.

As used herein, the phrase “therapeutically effective amount” means adose that provides the specific pharmacological effect for which thecompound or compounds are being administered. It is emphasized that atherapeutically effective amount will not always be effective inachieving the intended effect in a given subject, even though such doseis deemed to be a therapeutically effective amount by those of skill inthe art. For convenience only, exemplary dosages are provided below.Those skilled in the art can adjust such amounts in accordance withstandard practices as needed to treat a specific subject. Thetherapeutically effective amount may vary based on the route ofadministration and dosage form, the age and weight of the subject,and/or the subject's condition. For example one of skill in the artwould understand that the therapeutically effective amount for treatingdepression, anxiety, or PTSD may be different from the therapeuticallyeffective amount for treating acute exposure to a nerve agent. In thecontext of treating exposure to a nerve agent, the type and amount ofnerve agent exposure, and the amount of time that has passed betweenexposure and administration of a treatment may have a bearing on thedose needed to therapeutically effective.

The terms “treatment” or “treating” as used herein includes preventing,reducing, ameliorating, or eliminating one or more symptoms or effectsof the neurological condition being treating, or of exposure to a nerveagent.

The term “administering” as used herein includes prescribing foradministration as well as actually administering, and includesphysically administering by the subject being treated or by another.

As used herein “subject” or “individual” refers to any subject orindividual, such as a subject suffering from a neurological condition ora subject that has been exposed to or is at risk of being exposed to anerve agent, such as soman or sarin, and the terms are usedinterchangeably herein. In this regard, the terms “subject” and“individual” includes mammals, and, in particular humans.

As used herein, “child” generally refers to a human subject up to about18 years of age. As used herein, a child can be a subject who begins acourse of treatment prior to turning about 18 years of age, even if thesubject continues treatment beyond 18 years of age. In specificembodiments, a child may be less than 1 year old, less than 2 years old,less than 3 years old, less than 4 years old, less than 5 years old,less than 6 years old, less than 7 years old, less than 8 years old,less than 9 years old, less than 10 years old, less than 11 years old,less than 12 years old, less than 13 years old, less than 14 years old,less than 15 years old, less than 16 years old, less than 17 years old,or less than 18 years old.

Therapeutic Compounds

The disclosed methods are effective for treating or reducing the toxiceffects of exposure to a nerve agent and for treating neurologicalconditions, including epilepsy, various types of seizures, PTSD, SE,depression, and anxiety. The disclosed methods comprise administering(i) an AMPA/GluR5(GluK1) kainate receptor antagonist and (ii) an NMDAreceptor antagonist. The AMPA/GluR5(GluK1) kainate receptor antagonistmay be LY293558. Independently, the NMDA receptor antagonist may becaramiphen (CRM). In some embodiments, the methods further compriseadministering a positive allosteric modulator of synaptic GABA_(A)receptors, such as a benzodiazepine, such as midazolam, to the subject.

In subjects exposed to or at risk of exposure to a nerve agent, themethods may be effective to treat, prevent, reduce, ameliorate, oreliminate the effects of exposure to a nerve agent, such as seizures,brain damage, and the consequences on behavior, pathophysiologicalalterations in brain regions that underlie these deficits, and death. Insubjects suffering from a neurological condition, the methods may beeffect to treat, prevent, reduce, ameliorate, or eliminate the effectsof the neurological condition, such as seizures, brain damage,behavioral deficits, pathophysiological alterations in brain regionsthat underlie these deficits, and death.

The methods described herein exclusively target the glutamatergic systemwith at least two separate active agents, providing a new therapeuticapproach to treat exposure to nerve agents and neurological conditions;and may be particularly suitable for treating children. Indeed, themethods described herein represent the only therapy that has thepotential to fully succeed in treating both children and adultsintoxicated with nerve agents or suffering from other neurologicalconditions discussed herein. This is because, in the brain of a child,the GABAergic inhibitory system is not fully developed, and itsactivation (or facilitation, such as promoted by benzodiazepines such asdiazepam or midazolam) may produce depolarization and excitation insteadof inhibition. For similar reasons, the disclosed methods provide a newtherapeutic approach to treating, reducing the risks of, or preventingepilepsy, various types of seizures (e.g., seizures due to exposure to anerve agent, refractory seizures, seizures due to opiate withdrawal,etc.), PTSD, SE, depression, and anxiety. These conditions may also betreated with the disclosed therapeutic combinations in both children andadults. Thus, the methods described herein avoid those effects and socan be used to effectively treat nerve agent exposure and neurologicalconditions in both children and adults.

As noted above, the AMPA/GluR5(GluK1) kainate receptor antagonist may beLY293558 (also known as Tezampanel). LY293558((3S,4aR,6R,8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]decahydroisoquinoline-3-carboxylic acid) is an antagonist of theα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAreceptor) and kainate receptors containing the G1uR5 (GluK1) kainatesubunit (GluR5KRs).

As also noted above, the NMDA receptor antagonist may be caramiphen(CRM). Caramiphen (CRM) is an antagonist of the N-methyl-D-aspartate(NMDA) receptor with anticholinergic properties. CRM has a good safetyrecord in humans, including children. CRM was approved by the FDA in1973 as an over-the-counter antitussive for human use from the age oftwo years and above. The FDA approval was withdrawn in 1984 due to itslack of efficacy as an antitussive but not because of safety concerns.

While caramiphen may be used in some embodiments, other NMDA receptorantagonists also are suitable for use in the methods described herein,including other NMDA receptor antagonists with or without antimuscarinicactivity, including, but not limited to, APV(R-2-amino-5-phosphonopentanoate), AP7 (2-amino-7-phosphonoheptanoicacid), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonicacid), Selfotel, Amantadine, Atomoxetine, Agmatine, Chloroform,Dextrallorphan, Dextromethorphan, Dextrorphan, Diphenidine, Dizocilpine(MK-801), Ethanol, Eticyclidine, Gacyclidine, Ibogaine, Magnesium,Memantine, Methoxetamine, Nitromemantine, Nitrous oxide, Phencyclidine,Rolicyclidine, Tenocyclidine, Methoxydine, Tiletamine, Neramexane,Eliprodil, Etoxadrol, Dexoxadrol, WMS-2539, NEFA, Remacemide,Delucemine, 8A-PDHQ, Aptiganel (Cerestat, CNS-1102, HU-211,Rhynchophylline, and Ketamine.

Methods using both of these types of therapeutic compounds (anAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist) to treat exposure to nerve agents are particularlyadvantageous and show a synergistic increase in therapeutic effect ascompared to treatment with only one or the other. Thus, using both typesof therapeutic compounds permits the dose of each to be considerablylower than would be required if a compound were being used individually,thus decreasing the risks of side effects and increasing thetolerability of the treatment protocol in humans. In the discussion thatfollows, the AMPA/GluR5(GluK1) kainate receptor antagonist and NMDAreceptor antagonist are referred to collectively as the “therapeuticcompounds.” In some embodiments, the methods may further compriseadministering a positive allosteric modulator of synaptic GABA_(A)receptors, such as a benzodiazepine, such as midazolam, to the subject,in addition to the AMPA/GluR5(GluK1) kainate receptor antagonist andNMDA receptor antagonist (e.g., LY293558 and caramiphen, respectively).While not wanting to be bound by theory, it is believed thatbenzodiazepines such as midazolam exert anticonvulsant activity byenhancing inhibitory GABAergic transmission through an increase in thechannel opening frequency of the GABA_(A) receptors, a subsequentincrease in chloride conductance, and neuronal hyperpolarization.Midazolam is a benzodiazepine compound, and is a powerfulanticonvulsant, sedative, and anxiolytic compound, and a first-linetherapy for acute seizures and status epilepticus. It has a shorthalf-life and rapid onset of action when administered intravenously orintramuscularly to control acute repetitive seizures and SE. Midazolamhas an elimination half-life of 1.5-2.5 hours in adults, and a longerhalf-life in the elderly and young children. Midazolam may beadministered orally, buccally, nasally, intravenously, orintramuscularly.

Midazolam has been considered a better anticonvulsant than diazepam fornerve agent-induced seizures due to its improved water solubility whichprovides increased product stability and faster absorption. However,like diazepam, midazolam shows diminishing efficacy against nerveagent-induced seizures and SE with delayed administration (such asgreater than 40 minutes after nerve agent exposure).

Embodiments using therapeutic compounds from these three categories(such as LY293558, caramiphen and midazolam) will be particularlyefficacious because each exerts activity via a different mechanism ofaction, and so their use in combination may yield synergistic results.For example, in the context of treating, reducing the risks of, orpreventing seizures, including seizures induced by exposure to nerveagents, each exert anticonvulsant activity via a different mechanism ofaction, fostering a synergistic effect that will greatly reducehyperexcitability in the brain and effectively stop the seizures and SE.In addition, embodiments using therapeutic compounds from these threecategories will permit the dose of each compound to be considerablylower than would be required if a compound were being used individually,or in conjunction with only one other compound, thus decreasing therisks of side effects and increasing the tolerability of the treatmentprotocol in humans. In the discussion that follows, the term“therapeutic compounds” optionally includes a positive allostericmodulator of synaptic GABA_(A) receptors, such as a benzodiazepine, suchas midazolam, in addition to referring to an AMPA/GluR5(GluK1) kainatereceptor antagonist and NMDA receptor antagonist.

The methods described herein also are suitable for use in children.Although LY293558 has been shown to be effective in adult male andfemale rats exposed to soman (an organophosphorus nerve agent), forexample stopping seizures, protecting against neuronal damage, andpreventing the development of behavioral alterations when administered 1hour after exposure, it may not have the same effect in children. Thisis because, in the immature brain (such as in children) there is a highlevel of NMDA receptor activity, which, coupled with an augmentedglutamatergic tonus due to the OP-induced AChE inhibition, leads to anincreased Ca²⁺ influx through the NMDA channels. This is a primarymechanism for excitotoxicity or modifications and disfigurations of thedeveloping neuronal circuits. Moreover, the pediatric cholinergicsystem—which plays a central role in the mechanisms of nerve agentaction—is still at a developing stage in early postnatal life. Some ofthe differences between immature and adult animals results in lower AChEactivity in the prelimbic cortex, piriform cortex, and hippocampus ofthe immature animals, but not in the basolateral amygdala (BLA), whichplays a key role in seizure initiation after nerve agent exposure. Inaddition, differences in the blood-brain barrier permeability betweendeveloping and adult animals (e.g. humans) can affect thepharmacokinetics of injected drugs used to treat nerve agent exposure.Furthermore, developing animals differ from adult animals both inseizure susceptibility and in the extent and nature of neuropathologythat seizures can induce. Two particularly well-documented differencesbetween the developing brain (such as in children) and the adult brain:(1) In the developing brain, the GABAergic inhibitory system is stillweak, and its activation may produce depolarization and excitationinstead of inhibition. Therefore, facilitating the GABAergic system toprevent or halt seizures in the immature brain is unlikely to producethe desired results. (2) There is high NMDA receptor activity in theimmature brain, and it is well-known that Ca2+ influx through the NMDAchannels is a primary mechanism for excitotoxic death, or modificationsand disfigurations of the developing neuronal circuits.

The methods described herein address this by treating subjects with anNMDA receptor antagonist in addition to an AMPA/GluR5(GluK1) kainatereceptor antagonist. While not wanting to be bound by theory, it isbelieved that the NMDA receptor antagonist may protect the immaturebrain from seizure-induced damage. Thus, in the context of treatingchildren, the disclosed methods take into consideration theunderdeveloped GABAergic system in the young brain and the higheractivity of NMDA receptors therein. As such, the methods describedherein are suitable and effective for use in adults and children. Thiscontribution of the invention is particularly important in the contextof treating subjects exposed to or at risk of exposure to nerve agents,because immature animals (such as children) have a high propensity forseizures and are very susceptible to lethality after nerve agentexposure.

As discussed in more detail below, Examples 1-11, 16-19, and 25 discloseresults indicating that LY293558 either alone or in combination withcaramiphen and/or midazolam is capable of treating, reducing the risksof, or preventing seizures and other effects caused by exposure to nerveagents. The disclosed combination of therapeutic compounds allows for asynergistic means of treating exposure to nerve agents and reducing therisks of or preventing the toxic effects of such agents, given theunique mechanism of action of these compounds. Additionally, combiningall three disclosed types of therapeutic compounds will allow for asignificant reduction in the dose of the individual compounds comparedto when the compounds are administered alone.

As noted above, the disclosed methods using the disclosed combination oftherapeutic compounds, also are useful for treating adults and childrenwith neurological conditions including epilepsy, various types ofseizures (e.g., refractory seizures or seizures caused by chemical (e.g.alcohol or opiate) withdrawal), PTSD, SE, depression, and anxiety.

For example, epilepsy can arise from imbalances between excitatory andinhibitory synaptic transmission in key brain areas such as thehippocampus and temporal cortex, in which fast synaptic excitatoryneurotransmission is mediated via activation of ionotropic glutamatereceptor. Ionotropic glutamate receptor are divided on a molecular levelinto NMDA receptors, AMPA receptors, and kainate receptors. As notedabove, kainate receptors area further divided into multiple subtypes,and the GluR5 (GluK1) subtype is involved in the pathogenesis ofepilepsy. Indeed, antagonizing GluR5 (GluK1) has been shown to inhibitpilocarpine-induced and electrically evoked epileptiform activity, suchas seizures, in vitro and in vivo. NMDA receptor anatagonists are alsoeffective in antagonizing epileptiform activity. The disclosedcombination of an AMPA/GluR5 kainate receptor antagonist (such asLY293558) and NMDA receptor antagonist (such as caramiphen), optionallyin further combination with midazolam (or another benzodiazepine orpositive allosteric modulator of synaptic GABA_(A) receptors), isunexpectedly efficacious in treating, reducing the risks of, or preventepileptic activity, such as seizures—including complex partial seizuresand refractory seizures.

As discussed in more detail below, Examples 12 and 15 disclose resultsindicating that LY293558 alone is capable of treating, reducing therisks of, or preventing multiple types of seizures. The addition of anNMDA receptor antagonist and a positive allosteric modulator of synapticGABA_(A) receptors would be expected to allow for greater control andsuppression of seizures, given the unique mechanism of action of thesecompounds. Additionally, combining all three disclosed types oftherapeutic compounds will allow for a significant reduction in the doseof the individual compounds compared to when the compounds areadministered alone.

With regard to anxiety and depressive disorders, the amygdala, atemporal lobe structure that is part of the limbic system, has long beenrecognized for its central role in emotions and emotional behavior.Pathophysiological alterations in neuronal excitability in the amygdalaare characteristic features of certain psychiatric illnesses, such asanxiety disorders and depressive disorders. Furthermore, neuronalexcitability in the amygdala, and, in particular, excitability of thebasolateral nucleus of the amygdala (BLA) plays a pivotal role in thepathogenesis and symptomatology of temporal lobe epilepsy.

Thus, hyperexcitability of the amygdala is a characteristic ofdepression, anxiety disorders, and Posttraumatic Stress Disorder (PTSD).Hyperexcitability of the BLA is particularly noteworthy in theseconditions, and the remarkably high expression of GluK1 kainatereceptors (GluK1KRs) in the BLA may therefore serve as a target fortreatment. We have investigated the role of the GluK1KRs on themodulation of GABAergic and glutamatergic transmission in the BLA, andhave found that the net effect of GluK1KR activation is to increaseexcitability. Accordingly, blockade of GluK1KRs in the BLA hasanxiolytic effects, can therefore treat depression, anxiety, or PTSD.

Indeed, LY293558, as a GluK1KR/AMPA receptor antagonist, may produceanxiolytic effects in subjects with depression, anxiety, or PTSD.Moreover, caramiphen, blocks NMDA receptors and facilitates GABAergicinhibitory transmission and depressive patients are known to respond toNMDA receptor antagonists. Evidence in rats suggests that caramiphen, inparticular, has antidepressant effects. Therefore, the disclosedcombination of an AMPA/GluR5 receptor antagonist (such as LY293558) andNMDA receptor antagonist (such as caramiphen), optionally in furthercombination with midazolam (or another benzodiazepine or other positiveallosteric modulator of synaptic GABA_(A) receptors), may have superiorantidepressant and anxiolytic effects than the current standard of caretreatments.

Epilepsy and epileptic seizures that similarly have been shown to beassociated with neuronal excitability in the BLA, and the relevance ofthe GluR5KR function to epilepsy is suggested by the findings thatGluR5KR agonists can induce epileptic activity, whereas GluR5KRantagonists can prevent it. Further support for an important role ofGluR5KRs in epilepsy comes from the findings that antagonism of GluR5KRsis a primary mechanism underlying the antiepileptic properties of theanticonvulsant topiramate.

The disclosed combination of therapeutic compounds may also be usefulfor treating, reducing the risks of, or preventing seizures caused bywithdrawal from chemicals (e.g., alcohol or illicit drugs or opiates).Additionally, the disclosed combination of therapeutic compounds may beuseful for treating Neonatal Abstinence Syndrome (NAS) or reducing therisks of or preventing the long term psychological and physiologicaleffects of NAS. Indeed, antagonism of both AMPA and GluR5 receptors hasbeen shown to attenuate morphine-withdrawal-induced activation of locuscoeruleus neurons and behavioral signs of morphine withdrawal. In ananimal model of morphine withdrawal, pretreatment with LY293558significantly reduced the signs of morphine withdrawal in awake animals,including significant decreases in the occurrence of writhes, wet-dogshakes, stereotyped head movements, ptosis, lacrimation, salivation,diarrhea, and chews. We expect that adding an NMDA receptor antagonistto the treatment will improve the attenuation of withdrawal symptoms,including withdrawal-induced seizures, by modulating an alternativepathway that has also been implicated withdrawal-induced behavior andsymptoms.

As discussed in more detail below, Examples 13, 14, 23, and 24 discloseresults indicating that LY293558 alone is capable of treating symptomsof withdrawal (such as seizures) from alcohol, opiates and otherchemicals. The addition of an NMDA receptor antagonist and a positiveallosteric modulator of synaptic GABA_(A) receptors would be expected toallow for greater control and suppression of withdrawal-induced seizuresand other symptoms, given the unique mechanism of action of thesecompounds. Additionally, combining all three disclosed types oftherapeutic compounds will allow for a significant reduction in the doseof the individual compounds compared to when the compounds areadministered alone.

The present disclosure provides novel mechanisms for regulating neuronalexcitability in the BLA by modulating GABAergic inhibitory transmissionusing the disclosed combination of therapeutic compounds. Thesemechanisms involve the regulation of GABA release via both presynapticand postsynaptic kainate receptors containing the GluR5 (GluK1) subunit,by administering an AMPA/GluR5 kainate receptor antagonist (such asLY293558), and via NMDA receptors, by administering an NMDA receptorantagonist (such as caramiphen), optionally in further combination withmidazolam (or another benzodiazepine or another positive allostericmodulator of synaptic GABA_(A) receptors).This is particularly importantnot only in seizures and epilepsy, but also in anxiety disorders anddepression, which have been found to be associated with decreased GABAactivity in the BLA due to downregulation of α_(1A) adrenergicreceptors.

Pharmaceutical Compositions

The therapeutic compounds may be provided in the same or differentpharmaceutical compositions, which may include one or both of thetherapeutic compounds and a pharmaceutically acceptable carrier ordiluent. In embodiments where a positive allosteric modulator ofsynaptic GABA_(A) receptors, such as a benzodiazepine, such asmidazolam, also is being used, it may be provided in the samecomposition as one or both of the other therapeutic compounds, or in adifferent pharmaceutical composition.

The pharmaceutical compositions may be formulated for any route ofadministration, such as intravenous, subcutaneous, intraperitoneal,intramuscular, oral, nasal, pulmonary, ocular, vaginal, or rectaladministration. In some embodiments, one or both of the therapeuticcompounds are formulated for injection or infusion, such as beingformulated in a solution, suspension, emulsion, liposome formulation,etc. In some embodiments, one or both of the therapeutic compounds areformulated for oral administration, such as in a tablet, capsule,powder, granule, or liquid form suitable for oral administration. Thepharmaceutical compositions can be formulated to be immediate-releasecompositions, sustained-release compositions, delayed-releasecompositions, and combinations thereof, etc., using techniques known inthe art. In some embodiments, the therapeutic compounds are formulatedfor injection, such as intramuscular injection, and administered byinjection as a first treatment of exposure to a nerve agent. In someembodiments, the therapeutic compounds are formulated for oraladministration and administered orally to provide a subsequenttreatment, such as for maintenance therapy.

Suitable pharmacologically acceptable carriers and diluents for variousdosage forms are known in the art. For example, excipients, lubricants,binders, and disintegrants for solid preparations are known; solvents,solubilizing agents, suspending agents, isotonicity agents, buffers, andsoothing agents for liquid preparations are known. In some embodiments,the pharmaceutical compositions include one or more additionalcomponents, such as one or more preservatives, antioxidants, colorants,sweetening/flavoring agents, adsorbing agents, wetting agents and thelike.

Methods of Treatment

As noted above, the methods disclosed herein are effective for treatingor reducing the toxic effects of exposure to a nerve agent, comprisingadministering to a subject in need thereof (i) an AMPA/GluR5(GluK1)kainate receptor antagonist and (ii) an NMDA receptor antagonist, asdescribed above. In another aspect, the present disclosure providesmethods for treating, reducing the risks of, or preventing the effectsof neurological conditions, such as epilepsy, seizures (e.g., refractoryseizures, seizures caused by chemical (e.g. alcohol or opiate)withdrawal), post-traumatic stress disorder (PTSD), status epilepticus(SE), depression, or anxiety, comprising administering to a subject inneed thereof (i) an AMPA/GluR5(GluK1) kainate receptor antagonist and(ii) an NMDA receptor antagonist, as described above. In anyembodiments, the methods may further comprise administering atherapeutically effective amount of a positive allosteric modulator ofsynaptic GABA_(A) receptors, such as a benzodiazepine, such asmidazolam.

As noted above, the therapeutic compounds may be provided in a singlecomposition or in separate compositions, and may be administeredsubstantially simultaneously or sequentially with the compoundsadministered in any order. For example, the AMPA/GluR5(GluK1) kainatereceptor antagonist may be administered prior to the administration ofthe NMDA receptor antagonist, or the AMPA/GluR5(GluK1) kainate receptorantagonist may be administered after to the administration of the NMDAreceptor antagonist. Further, the positive allosteric modulator ofsynaptic GABA_(A) receptors (e.g., a benzodiazepine such as midazolam)may be administered prior to the AMPA/GluR5(GluK1) kainate receptorantagonist and the NMDA receptor antagonist, after the AMPA/GluR5(GluK1)kainate receptor antagonist and the NMDA receptor antagonist, or betweenthe AMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, with either the AMPA/GluR5(GluK1) kainate receptorantagonist or the NMDA receptor antagonist being administered first. Asused herein “substantially simultaneously” means that the compounds areadministered at the same time or within up to about 1 minute, about 2minutes, about 3, about 4, about 5, about 6, about 7, about 8, about 9,about 10, about 11, about 12, about 13, about 14, about 15, about 16,about 17, about 18, about 19, or about 20 minutes of one another.

As noted above, the therapeutic compounds may be administered by anysuitable route of administration, such as intravenously, subcutaneously,intraperitoneally, intramuscularly, orally, nasally, buccally,pulmonarily, ocularly, vaginally, or rectally. In some embodiments, thetherapeutic compounds are administered via injection, such asintramuscular injection. When the therapeutic compounds are administeredseparately, they may be administered via the same route ofadministration or different routes of administration. For example, theAMPA/GluR5(GluK1) kainate receptor antagonist may be administered viainjection (e.g. intramuscular injection) and the NMDA receptorantagonist may be administered orally, or vice versa. The positiveallosteric modulator of synaptic GABA_(A) receptors (e.g., abenzodiazepine such as midazolam) may be administered orally, buccally,nasally, intravenously, or intramuscularly, and it may be administeredvia the same route as either one or both of the AMPA/GluR5(G1uK1)kainate receptor antagonist and the NMDA receptor antagonist, or may beadministered via a different route than the AMPA/GluR5(GluK1) kainatereceptor antagonist and the NMDA receptor antagonist.

The methods may be used to treat or reduce the toxic effects of exposureto one or more nerve agents, including organophosphorus nerve agents andother chemicals that inhibit the activity of acetylcholinesterase.Exemplary, non-limiting examples of such nerve agents include sarin,soman, tabun, and cyclosarin. The methods may be used to treat, reducethe risks of or prevent the effects of neurological conditionscomprising epilepsy, seizures (e.g., refractory seizures, seizurescaused by chemical (e.g. alcohol or opiate) withdrawal), PTSD, SE,depression, or anxiety

One particular clinical benefit of the disclosed methods as compared toprior methods of treating exposure to nerve agents (i.e., administrationof diazepam) is that the methods described herein can be effective evenwhen the therapeutic compounds are administered at a later timepost-exposure. For example, in accordance with the methods describedherein, as subject may first be treated within up to 5 minutes, 10minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90minutes or up to 120 minutes after exposure. For example, in someembodiments, a subject is administered an AMPA/GluR5(GluK1) kainatereceptor antagonist and NMDA receptor antagonist within 20 minutes orless after exposure to the nerve agent, within one hour or less afterexposure to the nerve, or within two hours or less after exposure to thenerve agent.

As discussed above, the disclosed methods can be used to treat a rangeof subjects, including human and non-human animals, including mammals,as well as immature and mature animals, including human children andadults. In some embodiments, the subject has been exposed to a nerveagent, in other embodiments, the subject is suspected of having beenexposed to a nerve agent, while in other embodiments the subject is atrisk of exposure to a nerve agent. In some embodiments, the subject hasbeen diagnosed as having one or more neurological conditions, such asone or more of epilepsy, seizures (e.g., refractory seizures, seizurescaused by chemical or opiate withdrawal), PTSD, SE, depression, oranxiety. In other embodiments, the subject is suspected of having one ormore neurological conditions, while in other embodiments, the subject isat risk of developing one or more of neurological conditions.

As discussed above, the disclosed methods are effective to treat orreduce the risks of physiological consequences or toxic effects ofexposure to the nerve agent including, but not limited to, seizures,status epilepticus, brain damage, neurological effects, behavioraleffects, difficulty breathing, nausea, loss of control of bodilyfunctions, and death. Likewise, the disclosed methods can treatepilepsy, various types of seizures, PTSD, SE, depression, or anxiety,or decrease the symptoms or effects of any of these conditions.

In some embodiments, the method comprises administering a single dose ofthe therapeutic compounds (e.g., LY293558 and CRM), and, optionally, asingle dose of the positive allosteric modulator of synaptic GABA_(A)receptors (e.g., a benzodiazepine such as midazolam). In someembodiments administering a single dose of the therapeutic compounds iseffective to treat the exposure, i.e., to treat, reduce, ameliorate, oreliminate one or more of the physiological consequences of exposure, orto treat the neurological condition of the subject. In some embodiments,the method comprises administering repeated doses of one or more of thetherapeutic compounds, such as repeated doses of LY293558 and CRM,and/or repeated doses of midazolam, such as may be needed for one ormore symptoms or effects to be treated, reduced, ameliorated, oreliminated.

For instance, an individual that has been exposed to a nerve agent maybe evaluated for the presence and/or severity of signs and symptomsassociated with nerve agent intoxication, including, but not limited to,seizures, status epilepticus, brain damage, neurological effects,behavioral effects, difficulty breathing, nausea, or loss of control ofbodily functions, and may be treated with one or more of the therapeuticcompounds described herein until one or more of the signs/symptoms isreduced, ameliorated, or eliminated with treatment. Similarly, a subjectwith epilepsy, seizures, PTSD, depression, or anxiety may be evaluatedfor the presence and/or severity of signs and symptoms associated withthese conditions and may be treated with one or more of the therapeuticcompounds as described herein until one or more of the signs/symptoms isreduced, ameliorated, or eliminated. In some embodiments, treatment isrepeated with additional doses of one or more of the therapeuticcompounds if signs/symptoms/effects persist and can be continued (orrepeated) until one or more symptoms or effects of nerve agentintoxication are reduced, ameliorated, or eliminated.

In some embodiments, the method comprises administering periodic dosesof one or more of the therapeutic compounds, such as may be needed tomaintain recovery status after an initial treatment. That is, not onlyare the disclosed methods effective for treating or reducing the risk ofacute or recent exposure to a nerve agent, the disclosed methods alsocan be effective as maintenance therapy to maintain recovery statusand/or improve long-term outcome of subjects who have been exposed to anerve agent, or of subjects suffering from a neurological disorder.Maintenance therapy may comprise at least one, at least two, at leastthree, at least four, at least five, at least six, at least seven, atleast eight, at least nine, or at least ten subsequent administrationsof the therapeutic compounds (AMPA/GluR5(GluK1) kainate receptorantagonist and NMDA receptor antagonist) and, optionally, the positivemodulatory of synaptic GABA-A receptors (e.g., a benzodiazepine such asmidazolam). For the purposes of treating the neurological conditionsdisclosed herein, chronic maintenance therapy may be useful fortreating, reducing the risks of, or preventing, for example, epilepticseizures, depression, anxiety, or PTSD, while treating seizures arisingfrom chemical (e.g. alcohol or opiate) withdrawal may only requiretreatment for a limited period of time after detoxification has begun.

We have found that the lowest effective dose (LED) of LY293558,administered alone (i.m.), is 15 mg/kg, and that of caramiphen is 100mg/kg (i.m.). These doses are close to the median toxic doses (TD₅₀) forLY293558 (20 mg/kg) and caramiphen (162 mg/kg), respectively. Thus, thedisclosed combination therapy would be very beneficial as a treatmentbecause it would allow the doses of LY293558 and caramiphen to bereduced considerably, thus decreasing the incidence of side effects andincreasing the tolerability of the disclosed treatment in humans. Thisis because LY293558 and caramiphen produce their respectivepharmacological effect via different mechanisms of action, which fostersa synergistic effect for the disclosed methods of treatment. Based onthe pilot studies disclosed in the Examples, an effective dose ofLY293558 used in combination with caramiphen could be from about 2 toabout 5 mg/kg (i.m.), and an effective dose of caramiphen used incombination with LY293558 could be from about 10 to about 20 mg/kg(i.m.). As noted above, doses could be further reduced with a tripletherapy approach.

Thus, in the methods described herein, the dose of LY293558 may be fromabout 2 to about 20 mg/kg (i.m.), from about 2 to about 15 mg/kg (i.m.),or from about 2 to about 5 mg/kg (i.m.) when used in combination withcaramiphen, or less when used in combination with caramiphen andmidazolam. Thus, the dose of LY293558 may be about 0.025, about 0.05,about 0.075, about 0.1, about 0.25, about 0.5, about 0.75, about 1.0,about 0.25, about 0.5, about 0.75, about 1.0, about 1.25, about 1.5,about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0,about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about 4.5,about 4.75, about 5.0, about 5.25, about 5.5, about 5.75, about 6.0,about 6.25, about 6.5, about 6.75, about 7.0, about 7.25, about 7.5,about 7.75, about 8.0, about 8.25, about 8.5, about 8.75, about 9.0,about 9.25, about 9.5, about 9.75, about 10.0, about 10.25, about 10.5,about 10.75, about 11.0, about 11.25, about 11.5, about 11.75, about12.0, about 12.25, about 12.5, about 12.75, about 13.0, about 13.25,about 13.5, about 13.75, about 14.0, about 14.25, about 14.5, about14.75, about 15.0, about 15.25, about 15.5, about 15.75, about 16.0,about 16.25, about 16.5, about 16.75, about 17.0, about 17.25, about17.5, about 17.75, about 18.0, about 18.25, about 18.5, about 18.75,about 19.0, about 19.25, about 19.5, about 19.75, about 20.0, about21.0, about 21.25, about 21.5, about 21.75, about 22.0, about 22.25,about 22.5, about 22.75, about 23.0, about 23.25, about 23.5, about23.75, about 24.0, about 24.25, about 24.5, about 24.75, about 25.0 orgreater mg/kg (i.m.), or equivalent doses for a different route ofadministration.

In the methods described herein, the dose of caramiphen may be fromabout 10 to about 162 mg/kg (i.m.), or from about 10 to about 100 mg/kg(i.m.), or from about 10 to about 20 mg/kg (i.m.) when used incombination with LY293558, or less when used in combination withLY293558 and midazolam. Thus, the dose of caramiphen may be about 1.0,about 0.25, about 0.5, about 0.75, about 1.0, about 1.25, about 1.5,about 1.75, about 2.0, about 2.25, about 2.5, about 2.75, about 3.0,about 3.25, about 3.5, about 3.75, about 4.0, about 4.25, about 4.5,about 4.75, about 5.0, about 5.25, about 5.5, about 5.75, about 6.0,about 6.25, about 6.5, about 6.75, about 7.0, about 7.25, about 7.5,about 7.75, about 8.0, about 8.25, about 8.5, about 8.75, about 9.0,about 9.25, about 9.5, about 9.75, about 10.0, about 10.25, about 10.5,about 10.75, about 11.0, about 11.25, about 11.5, about 11.75, about12.0, about 12.25, about 12.5, about 12.75, about 13.0, about 13.25,about 13.5, about 13.75, about 14.0, about 14.25, about 14.5, about14.75, about 15.0, about 15.25, about 15.5, about 15.75, about 16.0,about 16.25, about 16.5, about 16.75, about 17.0, about 17.25, about17.5, about 17.75, about 18.0, about 18.25, about 18.5, about 18.75,about 19.0, about 19.25, about 19.5, about 19.75, about 20.0, about20.25, about 20.5, about 20.75, about 21.0, about 21.25, about 21.5,about 21.75, about 22.0, about 22.25, about 22.5, about 22.75, about23.0, about 23.25, about 23.5, about 23.75, about 24.0, about 24.25,about 24.5, about 24.75, about 25.0, about 25.25, about 25.5, about25.75, about 26.0, about 26.25, about 26.5, about 26.75, about 27.0,about 27.25, about 27.5, about 27.75, about 28.0, about 28.25, about28.5, about 28.75, about 29.0, about 29.25, about 29.5, about 29.75,about 30.0, about 31, about 32, about 33, about 34, about 35, about 36,about 37, about 38, about 39, about 40, about 41, about 42, about 43,about 44, about 45, about 46, about 47, about 48, about 49, about 50,about 51, about 52, about 53, about 54, about 55, about 56, about 57,about 58, about 59, about 60, about 61, about 62, about 63, about 64,about 65, about 66, about 67, about 68, about 69, about 70, about 71,about 72, about 73, about 74, about 75, about 76, about 77, about 78,about 79, about 80, about 81, about 82, about 83, about 84, about 85,about 86, about 87, about 88, about 89, about 90, about 91, about 92,about 93, about 94, about 95, about 100, about 101, about 102, about103, about 104, about 105, about 106, about 107, about 108, about 109.About 110, about 111, about 112, about 113, about 114, about 115, about116, about 117, about 118, about 119, about 120, about 121, about 122,about 123, about 124, about 125, about 126, about 127, about 128, about129, about 130, about 131, about 132, about 133, about 134, about 135,about 136, about 137, about 138, about 139, about 140, about 141, about142, about 143, about 144, about 145, about 146, about 147, about 148,about 149, about 150, about 151, about 152, about 153, about 154, about155, about 156, about 157, about 158, about 159, about 160, about 161,about 162 or greater mg/kg (i.m.), or equivalent doses for a differentroute of administration.

In the methods described herein, the dose of midazolam may be from about2 to about 10 mg/kg (i.m.). Thus, the dose of midazolam may be about2.0, about 2.25, about 2.5, about 2.75, about 3.0, about 3.25, about3.5, about 3.75, about 4.0, about 4.25, about 4.5, about 4.75, about5.0, about 5.25, about 5.5, about 5.75, about 6.0, about 6.25, about6.5, about 6.75, about 7.0, about 7.25, about 7.5, about 7.75, about8.0, about 8.25, about 8.5, about 8.75, about 9.0, about 9.25, about9.5, about 9.75, about 10.0, or equivalent doses for a different routeof administration.

As noted above, whenever multiple doses of the therapeutic compounds areadministered, they may be administered in the same or differentcompositions and by the same or different routes of administration as aprevious dose. For instance, in some embodiments, the initialadministration of the therapeutic compounds may be by intramuscularinjection and a subsequent administration be by oral administration.Alternatively, all administrations may be by intramuscular injection orall administrations may be orally. Or each therapeutic compound may beadministered by a different route.

EMBODIMENTS

The following is a list of non-limiting exemplary embodiments.

1. A method of treating or reducing the toxic effects of exposure to anerve agent, comprising administering to a subject in need thereof (i)an AMPA/GluR5(GluK1) kainate receptor antagonist and (ii) an NMDAreceptor antagonist.

2. A method of treating, reducing the risks of, or preventing aneurological condition, comprising administering to a subject in needthereof (i) an AMPA/GluR5(GluK1) kainate receptor antagonist and (ii) anNMDA receptor antagonist.

3. The method of embodiment 2, wherein the neurological condition isepilepsy, seizure, post-traumatic stress disorder, status epilepticus,depression, or anxiety.

4. The method of any one of embodiments 1-3, wherein theAMPA/GluR5(GluK1) kainate receptor antagonist is LY293558.

5. The method of any one of embodiments 1-4, wherein the NMDA receptorantagonist is an antimuscarinic compound.

6. The method of any one of embodiments 1-5, wherein the NMDA receptorantagonist is caramiphen.

7. The method of any one of embodiments 1-6, further comprisingadministering a positive allosteric modulator of synaptic GABA_(A)receptors to the subject.

8. The method of embodiment 7, wherein the positive allosteric modulatorof synaptic GABA_(A) receptors is a benzodiazepine.

9. The method of embodiment 7, wherein the positive allosteric modulatorof synaptic GABA_(A) receptors is midazolam.

10. The method of any one of embodiments 1-9, wherein theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, are administered in the same composition.

11. The method of any one of embodiments 1-9, wherein theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, are administered in separate compositions,by the same route of administration or by different routes ofadministration.

12. The method of embodiment 11, wherein the AMPA/GluR5(GluK1) kainatereceptor antagonist and the NMDA receptor antagonist , and, optionally,the positive allosteric modulator of synaptic GABA_(A) receptors, areadministered substantially simultaneously.

13. The method of embodiment 11, wherein the AMPA/GluR5(GluK1) kainatereceptor antagonist and the NMDA receptor antagonist, and, optionally,the positive allosteric modulator of synaptic GABA_(A) receptors, areadministered sequentially.

14. The method of embodiment 13, wherein the AMPA/GluR5(GluK1) kainatereceptor antagonist is administered prior to the administration of theNMDA receptor antagonist.

15. The method of embodiment 13, wherein the AMPA/GluR5(GluK1) kainatereceptor antagonist is administered after to the administration of theNMDA receptor antagonist.

16. The method of embodiment 13, wherein the positive allostericmodulator of synaptic GABA_(A) receptors is administered prior to boththe AMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist, after both the AMPA/GluR5(GluK1) kainate receptor antagonistand the NMDA receptor antagonist, or prior to one of theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist and after the other of the AMPA/GluR5(GluK1) kainate receptorantagonist and the NMDA receptor antagonist.

17. The method of any one of embodiments 1-16, wherein theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, are administered by injection.

18. The method of embodiment 17, wherein the injection is anintramuscular injection.

19. The method of any one of embodiments 1-16, wherein theAMPA/GluR5(GluK1) kainate receptor antagonist and the NMDA receptorantagonist and, optionally, the positive modulator of synaptic GABA-Areceptors, are administered orally.

20. The method of any one of embodiments 1 and 4-19, wherein the nerveagent comprises an organophosphorus toxin.

21. The method of any one of embodiments 1 and 4-19, wherein the nerveagent comprises soman.

22. The method of any one of embodiments 1 and 4-19, wherein the nerveagent comprises sarin.

23. The method of any one of embodiments 1 and 4-19, wherein the subjecthas been exposed to a nerve agent.

24. The method of embodiment 23, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist occurs within 20 minutes or less of exposure to the nerveagent.

25. The method of embodiment 23, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist occurs within one hour or less of exposure to the nerveagent.

26. The method of embodiment 23, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist occurs within two hours or less of exposure to the nerveagent.

27. The method of any one of embodiments 1 and 4-19, wherein the subjectis suspected of having been exposed to a nerve agent.

28. The method of any one of embodiments 1 and 4-19, wherein the subjectis at risk of exposure to a nerve agent.

29. The method of any one of embodiments 1 and 4-28, wherein the methodis effective to treat or reduce the toxic effects of exposure to thenerve agent selected from one or more of seizures, status epilepticus,brain damage, neurological effects, behavioral effects, difficultybreathing, nausea, loss of control of bodily functions, and death.

30. The method of any one of embodiments 3-19, wherein the seizure is arefractory seizure, an epileptic seizure, or a seizure induced bywithdrawal from a chemical (e.g. alcohol or opiate).

31. The method of embodiment 30, wherein the seizure is a seizureinduced by withdrawal from a chemical.

32. The method of embodiment 31, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, occurs prior to withdrawal of the chemical.

33. The method of embodiment 31, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist, and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, occurs during detoxification of thesubject.

34. The method of embodiment 31, wherein administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist, and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, occurs after withdrawal of the chemical.

35. The method of any one of embodiments 2-19, wherein the subject hasbeen diagnosed with the neurological condition.

36. The method of any one of embodiments 2-19, wherein the method iseffective to treat or reduce signs or symptoms of the neurologicalcondition.

37. The method of any one of the preceding embodiments, furthercomprising at least one subsequent administration of theAMPA/GluR5(GluK1) kainate receptor antagonist and NMDA receptorantagonist and, optionally, the positive allosteric modulator ofsynaptic GABA_(A) receptors, as maintenance therapy.

38. The method of embodiment 37, wherein the at least one subsequentadministration is administered by injection.

39. The method of embodiment 37, wherein the at least one subsequentadministration is administered orally.

40. The method of any one of the preceding embodiments, wherein thesubject is a mammal.

41. The method of any one of the preceding embodiments, wherein thesubject is a human child.

42. The method of any one of the preceding embodiments, wherein thesubject is a human adult.

43. An AMPA/GluR5(GluK1) kainate receptor antagonist and an NMDAreceptor antagonist, and, optionally a positive allosteric modulator ofsynaptic GABA_(A), as described herein, for treating or reducing thetoxic effects of exposure to a nerve agent, as described herein.

44. An AMPA/GluR5(GluK1) kainate receptor antagonist and an NMDAreceptor antagonist, and, optionally a positive allosteric modulator ofsynaptic GABA_(A), as described herein, for treating or reducing therisks of a neurological condition, as described herein.

45. Use of an AMPA/GluR5(GluK1) kainate receptor antagonist and an NMDAreceptor antagonist, and, optionally a positive allosteric modulator ofsynaptic GABA_(A), as described herein, in the preparation of one ormore medicaments as described herein for treating or reducing the toxiceffects of exposure to a nerve agent.

46. Use of an AMPA/GluR5(GluK1) kainate receptor antagonist and an NMDAreceptor antagonist, and, optionally a positive allosteric modulator ofsynaptic GABA_(A), as described herein, in the preparation of one ormore medicaments as described herein for treating or reducing the risksof a neurological condition as described herein.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples. Allprinted publications referenced herein are specifically incorporated byreference.

EXAMPLES

In the following experiments, unless otherwise stated, male and femalerats are used to detect any gender-related differences in the responsesto soman and/or the treatments. 21 day-old rats (P21) are used as modelsof 3-4-year old human children. 12-day old rats (P12) can be used asmodels of a newborn human.

Example 1—Combination Treatment of Adult Soman-Exposed Rats StopsSeizures

Glutamatergic excitation is reinforced during SE, not only because ofdisinhibition, but also because during SE there is up-regulation of AMPAreceptors, as well as enhanced expression of the GluK1 subunit, whichcould significantly contribute to worsening hyperexcitability andexcitotoxicity. Therefore, suppressing glutamatergic hyperactivity canbe a more effective way to control seizures, particularly if immediatetreatment is not possible and the treatment is administered with a delayafter seizure onset.

For example, LY293558 administered to adult rats 1 hour after exposureto soman stops seizures and fully protects against neuronal damage, andprevents increase in anxiety-like behavior and the associatedpathophysiological alterations in the basolateral amygdala (BLA).Similar results were obtained in 21 day-old rats (P21 rats). While notwanting to be bound by theory, in addition to suppressing AMPA receptoractivity, the efficacy of LY293558 may lie on the fact that GluK1Rs playan important role in the regulation of neuronal excitability of at leasttwo highly seizure-prone regions, the amygdala and the hippocampus. BothGABAergic and glutamatergic synaptic transmission are modulated byGluK1Rs in different brain regions, but the net effect of GluK1Ractivation appears to be an increase in excitability in both the BLA andthe hippocampus.

Caramiphen (CRM) is an M1 muscarinic receptor antagonist, but it hasalso been known to have antiglutamatergic properties. In the rat BLA,CRM antagonizes NMDA receptors. While not wanting to be bound by theory,this is probably because of its NMDAR antagonistic properties that CRMcan terminate nerve agent induced-seizures and provide neuroprotectionat latencies after exposure that are longer than those at which othermuscarinic antagonists (that do not have antiglutamatergic properties)are efficacious. When administered to adult rats, CRM displayed bothanti-seizure and neuroprotective effects even when administered at 30minutes or 60 minutes after soman exposure; however, it took 1 to 2hours for the behavioral seizure score to fall below stage 2.

Since CRM has pharmacological properties (NMDA antagonistic andantimuscarinic) that LY293558 does not have, a method using boththerapeutic compounds provides additional protection, as shown in thefollowing experiments.

Adult male rats were treated with LY293558 (15 mg/kg) and/or CRM (50mg/kg) after being exposed to 1.2×LD₅₀ soman (132 μg/kg; LD₅₀=110 μg/kgin adult rats). Electrodes were placed at various locations on the rats'brains (see FIG. 1C; 1: left frontal; 2: right frontal; 3: leftparietal; 4: right parietal; un-labeled: cerebellar reference electrode)to record electrical activity and assess the duration of initial SE andthe duration of SE during a 7-day period after exposure. FIG. 1A showsthe time course of seizure suppression by LY293558, while FIG. 1B showsthe time course of seizure suppression by LY293558+CRM, revealing afaster time-course of suppression by treatment of adult rats withLY293558+CRM. FIG. 1D shows a significantly shorter duration of bothinitial SE and total duration of SE during the 7 day period afterexposure in the LY293558+CRM group. FIG. 1D, left panel, shows theduration of the initial SE, which is the SE that started within 6-10 minafter soman exposure and was terminated by 15 mg/kg LY293558 (open bar),or 15 mg/kg LY293558 and 50 mg/kg CRM (closed bar). FIG. 1D, rightpanel, shows the duration of SE throughout the 7 day-period after somanexposure for the same two groups. The corresponding durations for theuntreated soman group (not shown) are about 700 min for the initial SEand more than 1000 min for the 7-day-period. *p<0.05 (Unequal Variancet-test). Thus, the combination treatment reduced the duration of theinitial SE and the total duration of SE as compared to treatment withLY293558 alone. Representative EEG tracings are shown in FIGS. 1A-1B,and additional, expended EEGs are shown in FIGS. 22A-22C.

Example 2—Combination Treatment of Adult Soman-Exposed Rats PreventsNeuronal Degradation

To understand whether LY293558 alone or in combination with CRM was moreeffective at preventing soman-induced neuronal degradation, adult ratswere exposed to 1.2×LD₅₀ soman (132 μg/kg) and then treated withLY293558 (15 mg/kg) and/or CRM (50 mg/kg) 20 minutes after somanexposure. Seven days after soman exposure, brain sections were taken andNissl-stained and scored or stained with Fluoro-Jade C. Results areshown in FIG. 2.

FIGS. 2A-D show that treatment with LY293558 and CRM is superior totreatment with LY293558 alone in preventing soman-induced neuronaldegeneration 7 days after exposure in adult rats. FIGS. 2A and 2B showpanoramic photomicrographs of Nissl-stained sections showing the brainregions from the Fluoro-Jade C photomicrographs shown in FIG. 2C. FIG.2C shows representative photomicrographs of Fluoro-Jade C stainedsections from the brain regions where neuronal degeneration wasevaluated, for the untreated (SOMAN), LY293558-treated (SOMAN+LY293558)and LY293558- and CRM-treated (SOMAN+LY293558+CRM) groups. Totalmagnification is 100×. Scale bar is 50 μm. FIG. 2D shows medianneuropathology score and interquartile range for the amygdala (Amy),piriform cortex (Pir), entorhinal cortex (Ent), the CA1 and CA3subfields of the ventral hippocampus, hilus, and neocortex (neo-Ctx).

Overall, this study shows that treatment with LY293558 and CRM iseffective at preventing neurodegeneration in all brain regions followingsoman exposure.

Example 3—Combination Treatment of Adult Soman-Exposed Rats PreventsWeight Loss

Adult rats were treated as outlined in Examples 1 and 2 and weighedevery day for seven days. Results are shown in FIG. 3. Soman-exposedrats that did not receive treatment (Soman, n=18) displayed dramaticweight loss and reduced weight gain. Soman-exposed rats treated withLY293558 only (n=18) exhibited significantly improved weight-gainresponse compared to the rats that did not receive the drug, whilesoman-exposed rats treated with both LY293558 and CRM (n=19) did notdisplay any difference from control rats (n=18) that were not exposed tosoman. Overall, this study shows that the combination of LY293558 andCRM is effective at preventing weight loss following soman exposure.

Collectively, the results of Examples 1-3 indicate that in adult ratsexposed to soman, treatment with both therapeutic agents (LY293558 andCRM) is superior to treatment with LY293558 alone, and provides morecomplete protection against soman-induced seizures, neurodegeneration.

Example 4—Combination Treatment of Immature Soman-Exposed Rats ImprovesBehavioral Outcomes

In this example, LY293558 and CRM were used to treat soman exposure in21 day old (P21) rats in order to obtain preclinical data on theefficacy of the combination therapy in a pediatric population.

P21 male and female rats were treated with LY293558 and/or CRM at 60minutes after 1.2×LD₅₀ (74.4 μg/kg; LD₅₀=62 μg/kg in immature rats)injection of soman. Seizure severity and duration were monitored, whileneuronal loss and degeneration, abnormal development (such as atrophy)of different brain regions, and/or the development of pathophysiologicaland behavioral deficits were determined at different time pointspost-exposure, as reflected in the data discussed below.

Referring to the protocol outlined in FIG. 4, P21 rats were exposed to1.2×LD₅₀ (74.4 μg/kg s.c.) of soman. Animals were treated with atropinesulfate (ATS) (0.5 mg/kg intramuscularly) and an acetylcholinesterasereactivator (HI-6) (125 mg/kg intraperitoneally) at 1 minutepost-exposure, for control of the peripheral cholinergic effects.Seizure onset was observed 5 minutes after exposures. The animals wereadministered LY293558 (15 mg/kg intramuscularly) and/or CRM (50 mg/kgintramuscularly) 60 minutes after soman exposure. Results are shown inFIGS. 4A-4B. As seen in those figures, treatment with both therapeuticcompounds, or with LY293558 alone, reduced development ofpathophysiological and behavioral deficits.

The Med Associates Acoustic Response Test System (Med Associates,Georgia, Vt.), which consists of a weight-sensitive platform inside anindividual sound-attenuating chamber containing a ventilated fan toprovide background noise, was used to assess the acoustic startleresponse. Movements in response to stimuli were measured as a voltagechange by a strain gauge inside each platform. Startle stimuli consistedof 110 or 120 dB sound pressure level noise bursts of 20-millisecondduration. Responses were recorded by an interfaced Pentium computer asthe maximum response occurring during the no-stimulus periods, andduring the startle period, and were assigned a value based on anarbitrary scale used by the software of the Test System. Results areshown in FIG. 4C.

A similar experiment was carried out in 12 day old rats, whichcorrespond to human newborns. P12 rats were exposed to 1.2×LD₅₀ (32.4μg/kg s.c.) of soman. Animals were treated with atropine sulfate (ATS)(0.5 mg/kg intramuscularly) and an acetylcholinesterase reactivator(HI-6) (125 mg/kg intraperitoneally) at 1 minute post-exposure, forcontrol of the peripheral cholinergic effects. Seizure onset wasobserved 5 minutes after exposures. The animals were administeredLY293558 (10 mg/kg intramuscularly) and/or CRM (50 mg/kgintramuscularly) 60 minutes after soman exposure. Results are shown inFIGS. 23A-23C, FIGS. 24A-24B, FIGS. 25A-25C, and FIGS. 26A-26B. As seenin those figures, treatment with both therapeutic compounds, or withLY293558 alone, reduced development of pathophysiological and behavioraldeficits at 30 days (FIGS. 23A-23C) and 3 months (FIGS. 25A-25C) postsoman exposure. These results additionally show that LY293558 alone orthe combination of LY293558 and caramiphen prevented the reduction incharge transferred by GABA_(A) receptor-mediated spontaneous IPSCs 30days after exposure to soman (FIGS. 24A-24B) and 3 months after somanexposure (FIGS. 26A-26B).

Considering the pronounced NMDA receptor activity in immatureanimals—which may be centrally responsible for the high seizuresusceptibility of the immature brain—as well as the role of NMDAreceptors in excitotoxicity, the benefit provided by treatment with bothLY293558 and CRM to immature animals is significant.

Example 5—Combination Treatment of Immature Soman-Exposed Rats ImprovesSurvival And Seizure Control

P21 rats were exposed to 1.2×LD₅₀ soman (74.4 μg/kg, s.c.) and treatedwith ATS (0.5 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) as outlined inExample 4. One hour after exposure, animals were administered LY293558(20 mg/kg, i.m.) and/or CRM (50 mg/kg, i.m.). Results are shown in FIG.5.

As shown in FIG. 5, animals treated with both therapeutic compoundsexhibited the highest 24-hour survival rates (95%) and seizure control(100%), with a pronounced reduction in latency to seizure control (13±4min compared to 25±3 min for LY293558 alone).

A similar experiment was performed in P12 rats, which correspond tonewborn human. P12 rats were exposed to 1.2×LD₅₀ soman (32.4 μg/kg,s.c.) and treated with ATS (0.5 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.)as outlined in Example 4. One hour after exposure, animals wereadministered LY293558 (10 mg/kg, i.m.) and/or CRM (50 mg/kg, i.m.).Results are shown in FIG. 21.

As shown in FIG. 21, newborn animals treated with both therapeuticcompounds exhibited 100% 24-hour survival rates and seizure control,with a pronounced reduction in latency to seizure control (18±5 mincompared to 35±6 min for LY293558 alone).

Example 6—Combination Treatment of Immature Soman-Exposed Rats ReducesSeizure Severity

P21 rats were exposed to 1.2×LD₅₀ soman (74.4 μg/kg, s.c.) and treatedwith ATS (0.5 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) as outlined inExample 4. One hour after exposure, animals were administered LY293558(20 mg/kg, i.m.) and/or CRM (50 mg/kg, i.m.). Results are shown in FIG.6.

As shown in FIG. 6, treatment with LY293558 and CRM reduces the severityof soman induced seizures faster than either compound alone in immaturerats. CRM alone, at a dose of 50 mg/kg, was not effective in reducingseizure severity within 120 min after exposure.

Collectively, Examples 4-6 show that in the P21 rats exposed to soman,treatment with both therapeutic agents was superior to treatment withLY293558 alone, as it stopped seizures significantly faster andincreased survival. This suggests that soman-induced damage of theamygdala and the hippocampus, as well as behavioral deficits, areprevented by the combination treatment.

Example 7—Combination Treatment of Immature Soman-Exposed Rats ReducesNeurodegeneration in the Amygdala

P21 rats were exposed to 1.2×LD₅₀ soman (74.4 μg/kg, s.c.) and treatedwith ATS (0.5 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) as outlined inExample 4. One hour after exposure, animals were administered LY293558(20 mg/kg, i.m.) and/or CRM (50 mg/kg, i.m.). Animals were monitored forup to 3 months: control animals (n=7), soman-exposed animals thatreceived CRM (50 mg/kg) at 60 minutes post-exposure (n=10),soman-exposed animals that received LY293558 (20 mg/kg) at 60 minutesafter soman-injection (n=10) and soman-exposed animals that receivedboth LY293558 and CRM at 60 minutes after soman-injection (n=10).

Degeneration of the amygdala was significantly reduced in animalstreated with both LY293558 and CRM, as shown in FIG. 7. For example,FIG. 7B shows group data of the estimated volume of amygdala for eachtreatment group, 30 days after the exposure (top) and 3 months after theexposure (bottom), with CRM alone failing to prevent degeneration of theamygdala.

Example 8—Combination Treatment of Immature Soman-Exposed Rats ReducesNeurodegeneration in the Hippocampus

P21 rats were exposed to 1.2×LD₅₀ soman (74.4 μg/kg, s.c.) and treatedwith ATS (0.5 mg/kg, i.m.) and HI-6 (125 mg/kg, i.p.) as outlined inExample 4. One hour after exposure, animals were administered LY293558(20 mg/kg, i.m.) and/or CRM (50 mg/kg, i.m.).

Animals were monitored for up to 3 months: control animals (n=7),soman-exposed animals that received CRM (50 mg/kg) at 60 minutespost-exposure (n=10), soman-exposed animals that received LY293558 (20mg/kg) at 60 minutes after soman-injection (n=10) and soman-exposedanimals that received both LY293558 and CRM at 60 minutes aftersoman-injection (n=10).

Degeneration of the hippocampus was significantly reduced in animalstreated with both LY293558 and CRM, as shown in FIG. 8. For example,FIG. 8B shows group data of the estimated volume of the hippocampus foreach treatment group, 30 days after exposure (top) and 3 months afterexposure (bottom), with CRM alone failing to prevent degeneration of thehippocampus.

Example 9—Acoustic Startle Response Testing in Immature Rats 30 DaysPost-Soman Exposure

P21 rats were treated according to the protocol outlined in Example 4and then assessed for acoustic startle response at 30 days post-somanexposure using the Med Associates Acoustic Response Test System outlinedin Example 4.

The results of this study showed that treatment with LY293558 and CRMreduced anxiety in open field and acoustic startle response tests 30days after exposure to soman, as shown by % time spent in the center ofthe open field, distance travelled, and Startle Response Amplitude, forthe control group (n=14), soman-exposed rats who received CRM (n=16),similarly treated rats who received LY293558 at 60 minutes after somanexposure (n=13), and rats who received both LY293558 and CRM (n=14).

Example 10—Acoustic Startle Response Testing in Immature Rats 3 MonthsPost-Soman Exposure

P21 rats were treated according to the outlined in Example 4 and thenassessed for acoustic startle response at 3 months post-soman exposureusing the Med Associates Acoustic Response Test System outlined inExample 4.

The results of this study showed that treatment with LY293558 and CRMreduced anxiety in open field and acoustic startle response tests 3months after exposure to soman, as shown by % time spent in the centerof the open field and distance travelled, and Startle ResponseAmplitude, for the control group (n=12), soman-exposed rats who receivedCRM (n=13), similarly treated rats who received LY293558 at 60 minutesafter soman exposure (n=13), and for rats who received combination ofLY293558 and CRM (n=12).

Results of Examples 9 and 10 show that treatment with both therapeuticcompounds significantly reduced anxiety (a marker of behavioral changes)at 30 days and 3 months post-exposure to soman, respectively.

Example 11—Electrophysiology Studies

P21 rats were treated according to the protocol outlined in Example 4and then electrophysiology studies were performed in in vitro brainslices (400 μm thick) containing the amygdala of the treated rats.

The slice medium consisted of (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄,21 NaHCO₃, 2 CaCl₂, 1 MgCl₂, and 11 D-glucose. For whole-cell recordingsof spontaneous inhibitory post-synaptic currents (IPSCs) from principalBLA neurons, the patch electrodes had resistances of 3.5-4.5 MΩ whenfilled with the internal solution: 60 mM CsCH₃SO₃, 60 mM KCH3SO3, 10 mMKCl, 10 mM EGTA, 10 mM HEPES, 5 mM Mg-ATP, 0.3 mM Na₃GTP (pH 7.2), 290mOsm.

Recorded currents were amplified and filtered (1 kHz) using the Axopatch200B amplifier (Axon Instruments, Foster City, Calif.). Field potentialswere evoked by stimulation of the external capsule at 0.05 Hz. Recordingglass pipettes were filled with ACSF (resistance ˜5 MΩ). For bothwhole-cell and field potential recordings, signals were digitized usingthe pClamp 10.2 software (Molecular Devices, Union City, Calif.).

Results are shown in FIG. 11, which indicate that treatment with boththerapeutic agents prevented a reduction in charge transferred byGABA_(A) receptor mediated spontaneous IPSCs 30 days (FIG. 11A) and 3months (FIG. 11B) post exposure to soman.

Collectively, the results of Examples 4-11 demonstrate the efficacy ofthe methods described herein in immature rats exposed to soman,indicating that the methods will be effective for treating childrenexposed to nerve agents. Treatment with both therapeutic agents(LY293558 and CRM) was superior to treatment with LY293558 alone,providing more complete protection against soman-induced seizures,neurodegeneration, and behavioral deficits, and also providing a veryrapid recovery from the toxic effects of exposure.

Example 12—Use of LY293558 in Treating AMPA Induced Seizures

LY293558 was evaluated in a mouse seizure model where seizures wereinduced by intracerebral injection ofS-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (S-AMPA, 1.25nmol), a dose and route known to induce seizures within minutes in90-100% of treated animals. LY293558 is also known to be an antagonistat the AMPA receptor and was administered intravenously at 1, 3, 10 and30 mg/kg 15 minutes prior to the S-AMPA and found to dose dependentlyreduce proportion of animals experiencing seizures with an ED50calculated to be 2.95 mg/kg.

The results indicate that LY293558 exhibits pharmacologic effects thatcould reduce seizures induced by neurotransmitters known to induceseizures. The addition of an NMDA receptor antagonist (e.g.,caramiphen), and, optionally, midazolam, to the treatment regimen couldfurther potentiate the observed pharmacological effects of LY293558.

Example 13—Use of LY293558 in Treating Opiate Withdrawal Symptoms

LY293558 was evaluated in a drug withdrawal model in morphine dependentrats. Morphine dependence was induced by subcutaneous implantation ofmorphine containing pellets daily for two days and allowing another 48hours for continual morphine absorption. Withdrawal was induced byremoving the morphine pellets followed by administration of the opioidantagonist naloxone. LY293558 was prophylactically administered (1, 10or 30 mg/kg, s.c.) at 15 minutes prior to precipitating withdrawal withthe naloxone. A panel of known withdrawal behaviors was scored over a 1hour observation period. A composite withdrawal score showed a dosedependent improvement in withdrawal symptoms.

Individual withdrawal symptoms such as ptosis, chewing, diarrhea,stereotyped head movements and writhing were all statisticallysignificantly reduced at the 10 mg/kg dose of LY293558. Additionalsymptoms of wet dog shakes, lacrimation and salivation weresignificantly reduced at the 30 mg/kg dose. Other symptoms of withdrawalincluding irritability, digging, jumping, chatter and erection were allnominally lower, but did not reach statistical significance. A parallelset of experiments evaluated neuronal activity in the locus coeruleus(LC) by in vivo electrophysiology in anesthetized animals. Morphinewithdrawal induced an 8-fold increase (0.5 Hz to 4 Hz) in LC neuronalcell firing, an effect that was dose-dependently inhibited by LY293558and nearly normalized (to 1 Hz) at the 10 mg/kg dose. The result issuggestive of LY293558 possessing beneficial effects for the adverse CNSconsequences of opiate withdrawal.

This model indicates that LY293558 can be effectively used in substanceabuse treatments, such as when LY293558 can be administered prior towithdrawal of the substance of abuse to alleviate and treat withdrawalsymptoms, and also indicates that LY293558 may be useful in treating orreducing withdrawal symptoms when administered after withdrawal of thesubstance of abuse. The addition of an NMDA receptor antagonist (e.g.,caramiphen), and, optionally, midazolam, to the treatment regimen couldfurther potentiate the observed pharmacological effects of LY293558.

Example 14—Use of LY293558 in Treating Seizures Induced by an IllicitDrug

LY293558 was evaluated in a catecholamine depleted rat model ofambulations induced by phencyclidine (also known as “angel dust” orPCP). In these studies, catecholamines were depleted for 24 hours priorto testing and ambulations were measured by breaks in photocell beams.Subcutaneous administration of PCP dramatically increased ambulatoryactivity (6× over controls). In animals pretreated with LY293558 (3mg/kg, sc) at 30 minutes prior to PCP administration, ambulations werereduced by 52%.

These results suggest glutamate receptor involvement in the ambulationsoccurring from exposure to PCP, and that LY293558 has activity inameliorating seizure-like activity arising from exposure to an illicitdrug of abuse. The addition of an NMDA receptor antagonist (e.g.,caramiphen), and, optionally, midazolam, to the treatment regimen couldfurther potentiate the observed pharmacological effects of LY293558.

Example 15—Use of LY293558 in Treating Seizures Induced by DirectElectrical and Additional CNS Stimuli

LY293558 was evaluated in a number of seizure models in both rats andmice where seizures were induced by a variety of stimuli includingelectrical shock (corneal electrode), pentylenetetrazole administration,audiogenic stimuli and amygdala stimulation. LY293558 was administeredprophylactically at a dose in the range of 0.3 to 30 mg/kg, s.c. at 30minutes prior to testing. LY293558 protected against tonic-clonicseizures occurring following 60 Hz (10 of 50 mA) of corneal electricalstimulation as well as the limbic seizures occurring with the 6 Hzstimulus. In these studies an ED50 for LY293558 was determined to be 9mg/kg, sc as a 30 minute pretreatment. In the pentylenetetrazolestudies, the proportion of mice completely protected from seizureincreased dose dependently by a 30 minute s.c. prophylactic pretreatmentwith LY293558 with an ED50 determined to be 10.3 mg/kg. When using anaudiogenic stimulus (120 dB white noise, 60 s) mice were dosedependently protected from tonic extensor seizures with an ED50 of 1.9mg/kg, s.c. as a 30 minute pretreatment. In contrast, LY293558 wasineffective in altering the threshold stimulus for evoking seizures whenusing direct stimulation of the amygdala via implanted electrodes.

These results suggest that LY293558 is effective at protecting fromseizures arising from a variety of triggers except for the amygdalakindling model where it may have been impossible for a pharmacologicagent to antagonize direct electrical stimulation to one of the seizurecenters of the brain (amygdala). The results also suggest that LY293558can reduce seizures triggered by either pharmacologic(phentylenetetrazole) or remote (corneal or audiogenic) stimulus, butnot when depolarizing currents are delivered directly to the amygdala.The addition of an NMDA receptor antagonist (e.g., caramiphen), and,optionally, midazolam, to the treatment regimen could further potentiatethe observed pharmacological effects of LY293558.

Example 16—Delayed LY293558 (1 Hour) Reduces Soman-Induced Mortality,Seizures and Neuropathology Without use of a Benzodiazepine

LY293558 was evaluated in a rat model of a G-series nerve agent, soman(GD), exposure. Soman was administered via a single subcutaneousinjection (154 mcg/kg, or 1.4×LD50. To increase survival rate, rats wereadministered HI-6(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropanedichloride; 125 mg/kg i.p.) at 30 minutes before soman exposure. HI-6 isa bispyridinium oxime (similar to pralidoxime) that reactivatesinhibited acetylcholinesterase. Within 1 minute after soman exposure,rats also received an intramuscular injection of atropine sulfate (2mg/kg) One hour after soman exposure, a group of rats was administeredLY293558 (50 mg/kg, intraperitoneal) and were compared to soman-exposedrats that received HI-6 and atropine but did not receive LY293558 (somanonly control arm). A subset group of animals was implanted withelectrodes for EEG documented seizure monitoring 1 week prior to somanexposure. Survival in non-EEG electrode animals for LY293558 recipientswas 100% (12 of 12) versus 55% (11 of 20) in the group that did notreceive LY293558 This difference in survival rate was statisticallysignificant (p<0.01; Pearson's chi-square and Friedman's exact test). Inanimals which had prior EEG electrode implants, survival was onlynominally higher in the LY293558 group (67%; 10 of 15) versus 54% in thecontrol group (6 of 11).

As shown in FIG. 12, the initial status epillepticus (SE) that developswithin 5-15 minutes following exposure to soman was dramatically reducedwith LY293558 vs. control group where SE continued for approximately 10hours before spontaneously resolving. When measured over a 24 hourperiod, total duration of SE was significantly reduced with LY293558.

Brains from a select group of animals that were maintained until either1 or 7 days post soman exposure were processed for histopathology withstaining by Nissl and Fluoro Jade C. As shown in FIG. 13, aneuropathology scoring system (0-4; as none, minimal, mild, moderate orsevere) was used to assess neuronal damage (cells staining positive withFJC) in subcortical regions of the brain in animals 7 days followingsoman exposure. The neuropathology score ranged from mild-to-moderate inregions studied. Treatment with LY293558 resulted in nominal protectionobserved in all regions, a finding that was statistically significant inmany regions of the subcortex. The addition of an NMDA receptorantagonist (e.g., caramiphen), and, optionally, midazolam, to thetreatment regimen could further potentiate the observed pharmacologicaleffects of LY293558

Example 17—Early LY293558 (20 Minute) Reduces Soman-Induced Mortality,Seizures and Neuropathology Without Use of a Benzodiazepine

LY293558 was evaluated in a rat model of a G-series nerve agent, soman(GD), exposure. Soman was administered via a single subcutaneousinjection (132 mcg/kg, which is approximately 1.2×LD50). To increasesurvival, twenty minutes after soman exposure, all rats received anintramuscular injection of 2 mg/kg atropine sulfate as well as anintraperitoneal injection of 125 mg/kg HI-6. In contrast to Example 16,in this study LY293558 was administered earlier (20 minutes from somanexposure vs. 1 hour), at a lower dose (15 mg/kg versus prior 50 mg/kg)and by an intramuscular route (vs. prior intraperitoneal). LY293558treated rats were compared to animals receiving soman exposure, atropineand HI-6.

Survival in non-EEG electrode animals for LY293558 recipients was 100%(18 of 18) versus 64% (18 of 28) in the group that did not receiveLY293558 This difference in survival rate was statistically significant(p<0.01; Pearson's chi-square).

As shown in FIG. 14, the initial status epilleptics (SE) that developswithin minutes following exposure to soman was dramatically reduced withLY293558 vs. control group where SE continued for approximately 10 hoursbefore spontaneously resolving. When measured over a 24 hour period,total duration of SE was significantly reduced with LY293558.

In this study, a small cohort of animals were reserved forpharmacokinetic assessment of LY293558. As shown in FIG. 15, LY293558 ismeasurable in the plasma and brain shortly (15 minutes) afterintramuscular administration and remain measurable to the final timepoint at 8 hours.

Brains from a select group of animals that were maintained until either1 or 7 days post soman exposure were processed for histopathology withstaining by Nissl and Fluoro Jade C. As shown in FIG. 16, aneuropathology scoring system (0-4; as none, minimal, mild, moderate orsevere) was used to assess neuronal damage (cells staining positive withFJC) in subcortical regions of the brain in animals 7 days followingsoman exposure. The neuropathology score ranged from mild-to-moderate inregions studied. Treatment with LY293558 resulted in statisticallysignificant neuroprotection all regions in the subcortex. The additionof an NMDA receptor antagonist (e.g., caramiphen), and, optionally,midazolam, to the treatment regimen could further potentiate theobserved pharmacological effects of LY293558.

Example 18—Comparison of the GluK1 Receptor Antagonist UBP302 VersusDiazepam in Soman-Induced Mortality, Seizures and Neuropathology

UBP302 ((S)-3-(2-carboxybenzyl)willardiine) is a glutamate GluK1receptor antagonist with an activity profile similar to LY293558 innerve agent seizure models. UBP302 was studied in a soman exposure modelwhere soman was administered via a single subcutaneous injection (154mcg/kg, or 1.4×LD50). To increase survival rate, HI-6 was administered(125 mg/kg i.p.) at 30 minutes before soman exposure. Within 1 minuteafter soman exposure, rats also received an intramuscular injection ofatropine sulfate. At either 1 or 2 hours post soman exposure, animalswere randomly selected to receive no further treatment (soman only arm),a 20 mg/kg intramuscular dose of diazepam (soman+DZP group) or a 250mg/kg intraperitoneal dose of UBP302 (soman+UBP group). Survival innon-EEG implanted animals was 63% (22 of 34) in the soman group andeither 91% (21 of 23) for diazepam or 96% (24 of 25) for UBP302 whenadministered at 1 hour post soman (p<0.02 for both groups versus somanonly group, Fischer exact test). The enhanced survival in both treatmentgroups suggests that either signaling through GABA-receptors withdiazepine or inhibition of glutamate signaling with UBP302 can confer asurvival benefit in this model

As shown in FIG. 17, the initial status epillepticus (SE) that developswithin minutes following exposure to soman was dramatically reduced withboth diazepam and UBP302 when the treatment agent is administered ateither 1 or 2 hours (upper panel, lower panel) following soman exposure.When measured over a 24 hour period, total duration of SE was notsignificantly reduced with diazepam whereas UBP302 was superior todiazepam in seizure control over 24 hours, achieving an approximate 65%suppression of seizure activity versus the soman only group. These dataindicate that seizure control by antagonizing glutamate receptors ismore effective at seizure suppression than stimulating GABA receptorsusing a benzodiazepine.

In animals that were maintained for 7 day histopathology, UBP302, butnot diazepam, administered 1 hour after soman exposure, reduced neuronaldegeneration in the subcortex. As shown in FIG. 18, the amygdala, CA1,and CA3 dorsal hippocampal areas, and entorhinal cortex had reducedneuronal degeneration in the subcortex 7 days after the exposure whenUBP302 was administered. The addition of an NMDA receptor antagonist(e.g., caramiphen), and, optionally, midazolam, to the treatment regimencould further potentiate the observed pharmacological effects ofLY293558.

Example 19—Comparison of Delayed (1 hour) LY293558 Versus Midazolam inSoman-Induced Mortality, Seizures, Behavioral and PathophysiologyAlterations 3 months After Soman

LY293558 was evaluated in a rat model of a G-series nerve agent, soman(GD), exposure. Soman was administered via a single subcutaneousinjection (132 mcg/kg, or 1.2×LD50). To increase survival rate, ratswere administered HI-6(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropanedichloride; 125 mg/kg i.p.) within 1 minute after soman exposure. HI-6is a bispyridinium oxime (similar to pralidoxime) that reactivatesinhibited acetylcholinesterase. Also, within 1 minute after somanexposure, rats received an intramuscular injection of atropine sulfate(2 mg/kg). One hour after soman exposure, a group of rats wasadministered LY293558 (15 mg/kg, i.m.) and another group received themidazolam (5 mg/kg i.m.). A subset group of animals was implanted withelectrodes, 1 week prior to soman exposure, for EEG-documented seizuremonitoring. Survival in non-EEG electrode-implanted animals for LY293558recipients was 92% (25 of 27) versus 96% (26 of 27) in the group thatreceived midazolam. This difference in survival rate was notstatistically significant (p>0.05; Friedman's exact test).

As shown in FIG. 19, the duration of the initial status epilepticus (SE;the SE that develops within 5-15 minutes following exposure to soman andis terminated after anticonvulsant treatment) was similar in theLY293558 and the midazolam groups. However, when measured over a 24-hourperiod post-exposure, total duration of SE was significantly lower inthe LY293558 group compared to the midazolam group (about 50% lower).

In animals that were maintained for 90 days after soman exposure,behavioral and pathophysiology tests showed that LY293558, but notmidazolam treatment, prevented increases in anxiety-like behavior andexaggerated startle responses, as well as reduction in GABAergicinhibitory currents in the basolateral amygdala nucleus, as shown inFIG. 20.

The addition of an NMDA receptor antagonist (e.g., caramiphen) to thetreatment regimen, and, optionally, the use of midazolam together withLY293558, could further potentiate the observed pharmacological effectsof LY293558.

Example 20—Clinical Studies of LY293558 in Humans

A clinical study was conducted to determine a maximum tolerated dose(MTD) in human volunteers beginning with an i.v. bolus dose of 0.01mg/kg. After an initial MTD was determined from n=6 volunteers the studywas expanded to an additional n=16 who received escalating doses todetermine an MTD for each individual. A total of 20 volunteers reached amaximum tolerated dose in a dose escalation protocol which escalated to2.0 mg/kg i.v. bolus. The adverse events were rated as mild in allinstances and include vision changes, sedation and headache. Visionchanges and sedation were noted beginning at 0.9 mg/kg and headache wasnoted in two individuals at 1.8 mg/kg.

Additionally, an efficacy study was conducted. The study was a blindedcrossover study of placebo vs. two doses of LY-293,558 at 33% and 100%of MTD where previous median MTD found to be 1.3 mg/kg. Twentyvolunteers received s.c. capsaicin injections in the volar surface ofthe forearm and spontaneous pain, allodynia and hyperalgesia wasassessed every 5 minutes for a 60 minute period. Spontaneous pain andallodynia were significantly reduced in both LY293558 groups vs.placebo. Pinprick hyperalgesia was nominally reduced at both doses andwas borderline significant p=0.05 at the highest dose. Mild visualsymptoms described as “looking through a haze” (or similar description)was noted in 19 of 20 volunteers (95%), and had resolved within an hourof onset. Additional side effects note included sedation (40%) andheadache (10%) and also resolved within an hour.

Example 21—Postoperative Dental Pain Study in Humans

This study was a placebo controlled study of 70 patients experiencingpain following dental surgery. The study compared two i.v. doses (0.4 of1.2 mg/kg) of LY293555 versus placebo or i.v. ketorolac (as a positivecontrol). Pain intensity was scored multiple times with a visual analogscale over a 240 minute period. Both LY293558 and ketorolacsignificantly reduced pain intensity compared to placebo. Incidence ofmild to moderate side effects was visual disturbance (20%), sedation(15%), headache (40%) and nausea (5%).

Example 22—Acute Migraine Study in Humans

This study was a placebo controlled study of 44 patients experiencingmoderate to severe migraine symptoms. The study compared i.v. LY293555(1.2mg/kg) versus a placebo or subcutaneous sumatriptin as a positivecontrol and evaluated symptoms for a 2 hour period. 54% of LY293558reported to be pain free at 2 hours versus 60% for sumatriptin and 6%for placebo. Similar response rates were reported for nausea,photophobia and phonophobia relief. Two patients (15%) of LY293558recipients experienced an adverse event including dizziness andsedation.

Example 23—Prophetic Alcohol and Opiate Withdrawal Phase II Trial

This example details a prospective phase II study in tremors/seizuresarising from drug withdrawal (either alcohol or opiates). Withdrawalsyndrome is preferred as the initial commercial indication because themarket is highly concentrated (specialized rehabilitation centers), thetiming of seizures can be anticipated (generally during first 72 hoursof detoxification), the overall clinical course is short (detox isgenerally complete in 1 week) and screening failures will be virtuallynon-existent (not enrolled until tremors/seizures begin). While alcoholis more widely used, opiate withdrawal has become a growing concern inthe U.S. The choice of which substance of abuse will be made afterfurther consultation with key opinion leaders in the area. A small phaseII study of a placebo and three dose-escalating treatment arms (40pts/arm) would be sufficient to demonstrate anticonvulsant activity inhumans. Such a study would entail administering an AMPA/GluR5(GluK1)kainate receptor antagonist (e.g., LY293558) and an NMDA receptorantagonist (e.g., caramiphen) to human subjects prophylactically,throughout detoxification, or some combination thereof in order toreduce, eliminate, or ameliorate at least one effect or symptom ofalcohol or opiate withdrawal, such as withdrawal-induced seizures.

Example 24—Prophetic Neonatal Abstinence Syndrome (NAS) Phase II Trial

This examples details a prospective phase II study in NAS. NAS is awithdrawal syndrome that occurs in newborns born to mothers who are drugaddicted (typically narcotics). According to recent CDC report and otherdata, the annual incidence is 21,000 cases per year, NAS represents upto 50% of all NICU stays, the incidence concentrates geographically(e.g., West Virginia and Vermont) as well as demographically (NativeAmericans). Withdrawal symptoms typically appear within the first 24-48hours postpartum and subside inside of a week. NAS may be moreattractive as an indication where successful attainment of pediatricrare disease designation and/or fast-track designation may speed theoverall approval of the commercial indication. A small phase II study ofa placebo and three dose-escalating treatment arms (15 neonates/arm)would be sufficient to demonstrate anticonvulsant activity in humans.Such a study would entail administering an AMPA/GluR5(GluK1) kainatereceptor antagonist (e.g., LY293558) and an NMDA receptor antagonist(e.g., caramiphen) to human infants having or suspected of having NASprophylactically, throughout the first 24-48 postpartum period, or somecombination thereof in order to reduce, eliminate, or ameliorate atleast one effect or symptom of NAS, such as neurological developmentdeficits.

Example 25—Prophetic Study of a Combination of LY293558, Caramiphen, andMidazolam to Treat Rats Exposed to Soman

This examples details a prospective preclinical study of treating ratswith a combination of LY293558, caramiphen, and midazolam after exposureto a nerve agent.

The study will have 3 specific aims. The first is to confirm thatLY293558 in combination with caramiphen (CRM) and midazolam (MDZ) iseffective in controlling sarin-induced seizures, in infant (P12), young(P21), adult (50 to 70 days-old), and aged (16 to 18 months-old) maleand female rats. To accomplish this aim will require determining theLD₅₀ of sarin for these ages and gender of rats, and confirming thatadministration of LY293558+caramiphen+midazolam, 30 min after sarinexposure (1.4×LD₅₀) will reduce or prevent lethality and suppress orblock seizures.

The second aim is to confirm that LY293558 in combination withcaramiphen and midazolam is effective in reducing or preventingsarin-induced neuronal degeneration and neuronal loss, in infant (P12),young (P21), adult (50 to 70 days-old), and aged (16 to 18 months-old)male and female rats. Neuronal degeneration will be assessed in a numberof seizure-prone brain regions, using FluoroJade-C staining. Neuronalloss will be assessed stereologically on Nissl-stained sections of theamygdala and the hippocampus. Neuropathology will be evaluated 1 day, 7days, 1 month, and 3 months after sarin exposure.

The third aim is to confirm that LY293558 in combination with caramiphenand midazolam is effective in reducing or preventing sarin-inducedpathophysiological alterations in the basolateral amygdala, as well asassociated behavioral deficits, in infant (P12), young (P21), adult (50to 70 days-old), and aged (16 to 18 months-old) male and female rats.Pathophysiology in the BLA will be assessed by studying excitabilityparameters in the BLA network, using whole-cell recordings in thevoltage-clamp and current-clamp mode, at 1 day, 7 days, 1 month, and 3months after sarin exposure. Two behavioral tests will be used to assessanxiety-like behavior, at 7 days, 1 month, and 3 months after sarinexposure. Correlations will be made between neuropathology,pathophysiology, and behavioral results.

The results from these studies will provide all the preclinicalinformation necessary to confirm that administration of this combinationcan be effectively and safely used as a treatment for nerve agentexposure, in an emergency situation, in order to protect the people,including the more vulnerable sections of the population.

Soman will be administered via a single subcutaneous injection (32μg/kg, which is 1.2×LD50). To increase survival, one minute after somanexposure, all rats will receive an intramuscular injection of 2 mg/kgatropine sulfate as well as an intraperitoneal injection of 125 mg/kgHI-6. Seizures generally develop in the first 10-15 minutes followingsoman administration. The general study schema appears in FIG. 27.

Neuropathological analysis will be performed at 30 days, 90 days, and 6months after soman exposure. All the procedures that will be used havebeen described extensively in the examples above.

Fixation & Tissue Processing. Rats will be deeply anesthetized withpentobarbital (75-100 mg/kg, i.p.) and transcardially perfused with PBS(100 ml) followed by 4% paraformaldehyde (200 ml). The brains will beremoved and post-fixed overnight at 4° C., then transferred to asolution of 30% sucrose in PBS for 72 hours, and frozen with dry icebefore storage at −80° C. until sectioning. Sections will be cutthroughout the rostrocaudal axis of the amygdala. One series of sectionswill be mounted on slides (Superfrost Plus, Daigger, Vernon Hills, Ill.)in PBS for Nissl staining with cresyl violet. Adjacent series ofsections will be stored at −20° C. in a cryoprotectant solution forGAD-67 immunohistochemistry. All neuropathological analysis will be donein a blind fashion.

Stereological Quantification. Design-based stereology will be used toquantify the total number of neurons in Nissl-stained sections in theBLA, at 1, 3, and 6 months after soman exposure. Sections were viewedwith a Zeiss Axioplan 2ie (Oberkochen, Germany) fluorescent microscopewith a motorized stage, interfaced with a computer runningStereolnvestigator 9.0 (MicroBrightField). The total number ofNissl-stained neurons will be estimated using the optical fractionatorprobe, and, along with the coefficient of error (CE), will be calculatedusing Stereo Investigator 9.0 (MicroBrightField). For Nissl-stainedneurons in the BLA, a 1-in-5 series of sections will be analyzed (8sections on average). An average of 365 neurons per rat will be counted.For GABAergic interneurons immuno-labeled for GAD-67 in the BLA (seeprocedure below), a 1-in-10 series of sections will be analyzed (onaverage 6 sections). An average of 260 interneurons per rat will becounted.

GAD-67 immunohistochemistry. To label GAD-67 immunoreactiveinterneurons, a 1-in-5 series of free-floating sections will becollected from the cryoprotectant solution, washed three times for 5 mineach in 0.1 M PBS, and then incubated in a blocking solution containing10% normal goat serum (NGS, Chemicon International, Temecula, Calif.)and 0.5% Triton X-100 in PBS for one hour at room temperature. Thesections will then be incubated with mouse anti-GAD67 serum (1:1000,MAB5406; Chemicon), 5% NGS, 0.3% Triton X-100, and 1% bovine serumalbumin, overnight at 4° C. After rinsing three times for 10 min each in0.1% Triton X-100 in PBS, the sections will be incubated withCy3-conjugated goat anti-mouse antibody (1:1000; Jackson ImmunoResearch,West Grove, PA) and 0.0001% DAPI (Sigma-Aldrich, St. Louis, Mo.) in PBSfor one hour at room temperature. After a final rinse in PBS for 10 min,sections will be mounted on slides, air dried for at least 30 min, andcoverslipped with ProLong Gold antifade reagent (Life Technologies,Grand Island, N.Y.).

Electrophysiology. Whole-cell recordings in the BLA of rats from the inthe BLA of rats from the Soman+LY293558+Caramiphen+Midazolam, andcontrol groups will be obtained at 1, 3, and 6 months after somanexposure. Coronal slices (400 μm) containing the BLA will be cut using avibratome (Leica VT 1200 S; Leica Microsystems, Buffalo Grove, Ill.), inice-cold cutting solution consisting of (in mM): 115 sucrose, 70 NMDG, 1KCl, 2 CaCl₂, 4 MgCl₂, 1.25 NaH₂PO₄, 30 NaHCO₃. Slices will betransferred to a holding chamber, at room temperature, in a bathsolution (artificial cerebrospinal fluid; ACSF) containing (in mM): 125NaCl, 3 KCl, 1.25 NaH₂PO₄, 21 NaHCO₃, 2 CaCl₂ 1.5 MgCl₂, and 20D-glucose (all purchased from Sigma-Aldrich, St. Louis, Mo.). Recordingsolution will be the same as the ACSF. All solutions will be saturatedwith 95% O₂, 5% CO₂ to achieve a pH near 7.4. Tight-seal (over 1 GQ)whole-cell recordings will be obtained from principal neurons in theBLA. Spontaneous inhibitory postsynaptic currents (sIPSCs) will berecorded at a holding potential of +30 mV. Ionic currents will beamplified and filtered (1 kHz) using the Axopatch 200B amplifier (AxonInstruments, Foster City, Calif.), digitally sampled (up to 2 kHz) usingthe pClamp 10.2 software (Molecular Devices, Sunnyvale, Calif.), andfurther analyzed using the Mini Analysis program (Synaptosoft Inc., FortLee, N.J.) and Origin (OriginLab Corporation, Northampton, Mass.).

Behavioral experiments. Animals from theSoman+LY293558+Caramiphen+Midazolam and control groups will be tested inthe open field and the acoustic startle apparatus, 1, 3 and 6 monthsafter soman exposure. In the open field apparatus (40×40×30 cm clearPlexiglas arena), anxiety-like behavior will be assessed as describedpreviously (Aroniadou-Anderjaska et al., 2012; Prager et al., 2014),following the procedure used by Grunberg and collaborators (Faraday etal., 2001). One day prior to testing, animals will be acclimated to theapparatus for 20 min. On the test day, the rats were placed in thecenter of the open field, and activity will be measured and recorded for20 min, using an Accuscan Electronics infrared photocell system(Accuscan Instruments Inc., Columbus, Ohio). Data are automaticallycollected and transmitted to a computer equipped with “Fusion” software(from Accuscan Electronics). Locomotion (distance traveled in cm), totalmovement time, and time spent in the center of the open field will beanalyzed.

Acoustic startle response (ASR) testing will be conducted with the useof the Med Associates Acoustic Response Test System (Med Associates,Georgia, Vt.), which consists of weight-sensitive platforms insideindividual sound-attenuating chambers. Each rat is individually placedin a ventilated holding cage. Each cage is placed on a weight-sensitiveplatform. Subjects' movements in response to stimuli will be measured asa voltage change by a strain gauge inside each platform. Startle stimuliwill consist of 120 or 110 dB sound pressure level noise bursts of 20-msduration. Responses will be recorded by an interfaced Pentium computeras the maximum response occurring during the no-stimulus periods andduring the startle period, and will be assigned a value based on anarbitrary scale used by the software of the test system.

Three months after soman-induced SE, animals from theSoman+LY293558+Caramiphen+Midazolam and control groups will be monitoredfor development of spontaneous recurrent seizures (SRS). The animalswill be video-monitored during the 3-month to the 6-month post-exposureperiod, because this is the time that the chronic phase of epilepsy(persistent SRS) is established after animals have experienced anepisode of prolonged SE (Amorin, B O et al. Epilepsy Behay. 2016 Jun.29; 61:168-173). The frequency of SRS, will be visually scored fromvideotapes obtained during recording sessions. SRS are characterized byclonic/tonic/tonic-clonic movements of the forelimbs culminating withrearing and falling (stages 3 to 5 according to Racine 1972).

These experiments will confirm the efficacy of the combination therapy(LY293558 administered in combination with caramiphen and midazolam) inpreventing neuropathological, pathophysiological, behavioral, andneurological deficits at 30 days, 90 days and 6 months after exposure ofadult male rats to soman. At these time points, correlations will bemade of anxiety-like behavior and recurrent epileptic seizures with theneuronal and GABAergic interneuronal loss in the BLA, as well as withthe level of spontaneous/background GABAergic inhibition in the BLA, abrain region that plays a key role in both emotional behavior andseizure generation.

1. A method of treating or reducing the toxic effects of exposure to anerve agent, or treating or reducing the risks of a neurologicalcondition, comprising administering to a mammalian subject in needthereof: (i) an AMPA/GluR5(GluK1) kainate receptor antagonist and (ii)an NMDA receptor antagonist, and, optionally, further comprisingadministering a positive allosteric modulator of synaptic GABAAreceptors to the subject, wherein the AMPA/GluR5(GluK1) kainate receptorantagonist and the NMDA receptor antagonist and, optionally, thepositive allosteric modulator of synaptic GABA.sub.A receptors, areadministered orally or via a route of injection selected fromintravenous, subcutaneous, and intraperitoneal.
 2. (canceled)
 3. Themethod of claim 1, wherein the method is for treating a neurologicalcondition selected from epilepsy, seizure, post-traumatic stressdisorder, status epilepticus, depression, and anxiety.
 4. The method ofclaim 1, wherein the AMPA/GluR5(GluK1) kainate receptor antagonist isLY293558.
 5. The method of claim 1, wherein the NMDA receptor antagonistis an antimuscarinic compound.
 6. The method of claim 1, wherein theNMDA receptor antagonist is caramiphen.
 7. The method of claim 1,wherein the optional positive allosteric modulator of synaptic GABAAreceptors is administered.
 8. The method of claim 7, wherein thepositive allosteric modulator of synaptic GABAA receptors is abenzodiazepine.
 9. The method of claim 7, wherein the positiveallosteric modulator of synaptic GABAA receptors is midazolam.
 10. Themethod of claim 1, wherein the AMPA/GluR5(GluK1) kainate receptorantagonist is LY293558 and the NMDA receptor antagonist is caramiphen,and the optional positive allosteric modulator of synaptic GABAAreceptors, if administered, is midazolam. 11-17. (canceled)
 18. Themethod of claim 1, wherein the AMPA/GluR5(GluK1) kainate receptorantagonist and the NMDA receptor antagonist and, optionally, thepositive allosteric modulator of synaptic GABAA receptors, areadministered by a route of injection selected from intravenous,subcutaneous, and intraperitoneal.
 19. (canceled)
 20. The method ofclaim 1, wherein the AMPA/GluR5(GluK1) kainate receptor antagonist andthe NMDA receptor antagonist and, optionally, the positive modulator ofsynaptic GABA-A receptors, are administered orally.
 21. The method ofclaim 1, wherein the method is for treating or reducing the toxiceffects of exposure to a nerve agent that comprises an organophosphorustoxin.
 22. The method of claim 1, wherein the method is for treating orreducing the toxic effects of exposure to a nerve agent that comprisesone or more selected from soman and sarin.
 23. The method of claim 1,wherein the method is for treating or reducing the toxic effects ofexposure to a nerve agent and the subject has been exposed to the nerveagent. 24-26. (canceled)
 27. The method of claim 1, wherein the methodis for treating or reducing the toxic effects of exposure to a nerveagent and the subject is suspected of having been exposed to a nerveagent or is at risk of exposure to a nerve agent.
 28. The method ofclaim 1, wherein the method is effective to treat or reduce the toxiceffects of exposure to the nerve agent selected from one or more ofseizures, status epilepticus, brain damage, neurological effects,behavioral effects, difficulty breathing, nausea, loss of control ofbodily functions, and death. 29-40. (canceled)
 41. The method of claim1, wherein the subject is a human adult. 42-45. (canceled)